Mammalian relaxin receptors

ABSTRACT

High affinity relaxin receptors, polypeptide compositions related thereto, as well as nucleotide compositions encoding the same, are provided. These proteins, herein termed LGR7 and LGR8, are orphan leucine-repeat-containing, G protein-coupled receptors. These receptors have a wide and a unique tissue expression pattern. The receptors, particularly soluble fragments thereof, are useful as therapeutic agents capable of inhibiting the action of relaxin and InsL3. The receptors and fragments thereof also find use in the screening and design of relaxin agonists and antagonists. Conditions treatable with relaxin agonists or antagonists include prevention or induction of labor, treatment of endometriosis, treatment of skin conditions such as scleroderma that require collagen or extracellular matrix remodelling. Additionally, relaxin has been implicated in the dilation of blood vessels&#39; smooth muscle cells directly and through release of nitric oxide and atrial natriuretic peptide. Relaxin has also been used in the treatment of severe chronic pain, particularly pain arising from stretching, swelling, or dislocation of tissues.

BACKGROUND OF THE INVENTION

[0001] Relaxin is a pregnancy hormone discovered in 1926 (Hisaw (1926)Proc. Soc. Exp. Biol. Med. 23: 661-663), based on its ability to relaxthe public ligament in guinea pig. Mature human relaxin is a hormonalpeptide of approximately 6000 daltons known to be responsible forremodelling the reproductive tract before parturition, thus facilitatingthe birth process. A concise review of relaxin was provided by Sherwood,D. in The Physiology of Reproduction, Chapter 16, “Relaxin”, Knobil, E.and Neill, J., et al. (eds.), (Raven Press Ltd., New York), pp. 585-673(1988). Relaxin has local autocrine and/or paracrine roles thatcontribute to connective tissue remodeling at the maternal-fetalinterface during late pregnancy and at parturition, including anincrease in the expression of the genes, proteins, and enzyme activitiesof the matrix metalloproteinases interstitial collagenase (MMP-1),stromelysin (MMP-3), and gelatinase B (MMP-9).

[0002] Two human gene forms of relaxin have been identified, (H1) and(H2) (Hudson et al. (1983) Nature 301:628-631; Hudson et al. (1984) EMBOJournal 3:2333-2339; U.S. Pat. Nos. 4,758,516 and 4,871,670). Only theH2 form is expressed in corpus luteum. The primary translation productof H2 relaxin is a preprorelaxin consisting of a 24 amino acid signalsequence followed by a B chain of about 29 amino acids, a connectingpeptide of 104-107 amino acids, and an A chain of about 24 amino acids.

[0003] Although relaxin itself has been well-characterized for a numberof years, it's receptor has remained elusive. To date, binding studieshave had to rely on crude cellular extracts, which indicated that aspecific binding molecule was present, but gave no clue as to itsmolecular identity. Relaxin binding sites have been reported in thereproductive tract (Kohsaka et al. (1998) Biol Reprod 59(4):991-9), aswell as other tissues, including cardiac and other smooth muscle, andspecific nuclei in the brain (Tan et al. (1999) Br J Pharmacol127(1):91-8).

[0004] During fetal development, the sexual dimorphic position of thegonads in mammals is dependent on the differential development of twoligaments. In males, growth of the gubernaculum and regression of thecranial suspensory ligament results in transabdominal descent of thetestes. Impaired testicular descent (cryptorchidism) is a prevalentcongenital abnormality in humans, found in 2% of male births. INSL3,also known as Leydig insulin-like peptide or relaxin-like factor (RLF),is one of the seven relaxin-like genes in humans known to be expressedin Leydig cells of fetal and adult testes as well as in theca and lutealcells of the postnatal ovary (Ivell (1997) Rev. Reprod. 2, 133-8). Malemice mutant for INSL3 exhibit bilateral abdominal cryptorchidism whereasfemale mice overexpressing INSL3 showed ovary descent and displayedbilateral inguinal hernia. Although INSL3 binds to gubernacularhomogenates (Boockfor et al. (2001) Reproduction 122, 899-906) andinduces growth of rat gubernaculum in whole organ cultures (Smith et al.(2001) J. Pept. Sci. 7, 495-501), the exact nature of the INSL3 receptoris unknown. A recent study indicated that transgene integration in crspmice resulted in a 550-kb deletion located upstream of the Brca2 gene,leading to defective testis descent (Overbeek et al. (2001) Genesis 30,26-35).

[0005] The identification and molecular characterization of relaxinreceptors is of great scientific and clinical interest. Understanding ofrelaxin signaling mechanisms mediated by its receptor can provide newapproaches for the regulation of relaxin target tissues during pregnantand non-pregnant states.

SUMMARY OF THE INVENTION

[0006] High affinity relaxin receptors, polypeptide compositions relatedthereto, as well as nucleotide compositions encoding the same, areprovided. These proteins, herein termed LGR7 and LGR8, are orphanleucine-repeat-containing, G protein-coupled receptors, and are paralogsof gonadotropin and thyrotropin receptors. These receptors have a wideand unique tissue expression pattern. LGR8 is a receptor for InsL3,which has been shown to be important for testis descent. Treatment ofantepartum animals with the soluble ligand-binding region of LGR7 hasled to parturition delay. Thus, relaxin and InsL3 signal through Gprotein-coupled receptors distinct from the related insulin family ofligands in regulating pregnancy-related processes.

[0007] The receptors, particularly soluble fragments thereof, are usefulas therapeutic agents capable of inhibiting the action of relaxin. Thereceptors and fragments thereof also find use in the screening anddesign of relaxin agonists and antagonists. Conditions treatable withrelaxin agonists or antagonists include prevention or induction oflabor, treatment of endometriosis, treatment of skin conditions such asscleroderma that require collagen or extracellular matrix remodelling.Additionally, relaxin has been implicated in the dilation of bloodvessels' smooth muscle cells directly and through release of nitricoxide and atrial natriuretic peptide. Relaxin has also been used in thetreatment of severe chronic pain, particularly pain arising fromstretching, swelling, or dislocation of tissues.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1. Cloning of LGR8 and elucidation of its signalingmechanism. A) Sequence alignment of human LGR8 (Accession No. AF403384)with homologous receptors belong to the same subgroup of LGRs (humanLGR7, a snail LGR and a Drosophila LGR). The sequence identity of humanLGR8 was first deduced from high throughput genomic sequences using geneprediction programs GRAIL-1.3, FGENES-M, and NNPP located at BCMLauncher server, followed by subcloning using 5′ and 3′ RACE usinggene-specific primers and Marathon-ready cDNA template (Clontech Inc.,Palo Alto, Calif.) from human ovary, testis, pituitary, brain, anduterus. Amino acid numbers are on the right and the stop codon is markedwith an asterisk. Putative N-glycosylation sites are circled. Identicalresidues are highlighted by a dark background. The N-terminal signalpeptide for secretion in each polypeptide is underlined. The consensusLDL receptor cysteine-rich motifs are indicated by bold italics.Different structural motifs including the transmembrane (TM) region,intracellular loop (IL), and extracellular loop (EL), are indicated byarrowheads. Shaded residues are identical in the human LGR7 and snailLGR sequences. Residue numbers are shown on the right and gaps areincluded for optimal protein alignment. The BLOCK Maker program was usedto align and generate the highly conserved ungapped blocks of thealigned LGR polypeptides from different species. B) Phylogeneticrelatedness of diverse LGRs from mammals and invertebrates. Full-lengthamino acid sequences of 11 LGRs from mammals (LH, FSH, and TSH receptorsplus LGR4 to LGR8), sea anemone, nematode, pond snail, and Drosophilawere analyzed by the neighboring-joining method from the Blockalignments using a routine in CLUSTALW. h: human; s: pond snail; Dm:Drosophila melanogaster analyzed using the Blocks program. These LGRscould be divided into three major branches; the first subgroup includethe three classical human glycoprotein hormone receptors as well as LGRsfrom sea anemone, nematode, and Drosophila, the second group includeshuman LGR4-6 and Drosophila LGR2, whereas the third subgroup includeshuman LGR7-8 and Drosophila LGR3. C) A gain-of-function LGR8 mutantmediated constitutive cAMP production in transfected cells. Based on thegain-of-function point mutation (LHR D578Y) found in the LH receptorgene of patients with familial male-limited precocious puberty, LGR8with a homologous point mutation in transmembrane VI (LGR8 D637G) wasgenerated. After transfection of expression constructs encodingwild-type or mutant receptors into 293T cells, basal cAMP levels weremonitored. Transfection of 293T cells with increasing concentrations(0-500 ng/well) of expression vectors encoding LGR8 D647Y, LGR7(1) D637Yand LH receptor D578Y led to increases in basal cAMP levels intransfected cells. In contrast, cAMP levels in cells transfected withwild-type (WT) receptors were negligible (n=3; mean±SE).

[0009]FIG. 2. LGR7 and LGR8 are relaxin receptors. Porcine relaxinstimulated cAMP production by 293T cells expressing recombinant LGR7 (A)and LGR8 (B). Relaxin, but not insulin, IGF-1, or IGF-IIdoes-dependently increased the cAMP production by 293T cellsoverexpressing recombinant receptors. In addition, glucagon, aGs-coupled receptor activator, also has no effect on cAMP production byLGR7 and LGR8. Although there are two splicing variants for human LGR7(Hsu et al. (2000) Mol Endocrinol. 14(8):1257-71), the long form wasused exclusively for the present analysis.

[0010]FIG. 3. Tissue distribution of LGR7 and LGR8. A) Profiles of LGR7and LGR8 transcripts in human tissues. PCR analyses of LGR7 and LGR8 indiverse human tissues were conducted using human cDNA from ovary,testis, kidney, thyroid, spleen, brain, pancreas, pituitary, uterus,prostate, heart, hypothalamus, placenta, and lymph nodes (1 ug/reaction)and LGR gene-specific primer pairs under high-stringency conditions.Specific bands are indicated by arrowheads. PCR amplification wascarried out under high stringency conditions to minimize nonspecificsignals (denaturation: 94C, 30 sec, annealing and extension: 68-72C, 3min; 35 cycles). B) Immunohistochemical analysis of LGR7 expression inrodent reproductive tracts. Specific LGR7 staining in uterus, cervix,mammary gland, and pituitary were indicated by black arrowheads.Epithelial layer (EL); stromal layer (SL); Muscularis layer (ML);mammary gland and nipple, anterior pituitary, intestine, skin, etc.Staining using control antiserum showed negligible signals.

[0011]FIG. 4. Neutralization of relaxin actions using the ligand bindingdomain of LGR7. A) Generation of the soluble ectodomain of LGR7 (7BP)using an anchored receptor approach. Permanent 293T cell linesoverexpressing 7BP-CD8 were cultured under serum-free conditioned andtreated with thrombin for three days, and soluble recombinant 7BP taggedwith 6-His and M1 epitopes was purified using Nickel and anti-FLAGaffinity chromatography under natural conditions. Specific 7BP bandsfollowing Western blotting analysis using M1-FLAG antibody was indicatedby an arrowhead (lane B). Homologous domain of LGR4 (4BP) was alsogenerated using the same approach and shown on lane C. B) Specificinteraction of relaxin and 7BP. Purified porcine relaxin was incubatedwith recombinant 7BP in PBS and crosslinked with disuccinimidyl suberat,before boiling for 5 min. under denaturing conditions and resolved in7.5% SDS PAGE. The relaxin/7BP complex was detected by an anti-relaxinantibody whereas 4BP showed negligible interaction with relaxin. C)Recombinant 7BP blocked the stimulatory effects of relaxin on LGR7 andLGR8. 293T cells expressing LGR7 or LGR8 were treated with 0.1 nMrelaxin in combination with different dosage of 7BP or 4BP for 24 hunder serum-free conditions. D) Treatment of cultured rat myometrialcells with 7BP blocked the relaxin stimulation of cAMP production.Uterine tissues were obtained from 25-day-old female rats implanted withdiethystibestrol for 3 days. Myometrial cells were prepared by serialdigestions with trypsin and collegenase and cultured. Cells were treatedwith 1 nM of porcine relaxin with or without recombinant 7BP underserum-free conditions for 24 h.

[0012]FIG. 5. Activation of LGR8 but not LGR7 by INSL3. Cells expressingrecombinant human LGR8 or LGR7 were treated with INSL3 from differentspecies, or with biotinylated-ovine INSL3 (Biotin-INSL3), porcinerelaxin (RLX) or glucagon. Ligand signaling was estimated based onextracellular cAMP production. A) LGR8. B) LGR7.

[0013]FIG. 6. Direct binding of biotinylated INSL3 to LGR8. A) Ligandbinding assays. Cells expressing LGR8 were incubated with 5 nM ofbiotinylated ovine INSL3 with or without increasing levels of rat INSL3.Specific INSL3 binding to LGR8 was estimated using labeled strepavidin.B) Cross-linking of INSL3 to LGR8. Cells expressing LGR8 were incubatedwith biotinylated INSL3 (Biotin-INSL3) with or without a 20-fold excessINSL3 before cross-linking. Complexes of biotinylated INSL3 and LGR8were detected using the avidin-HRP following SDS-PAGE and proteinblotting. Lane 1, biotin-INSL3 alone; lane 2, INSL3-LGR8 complexes; lane3; competition with excess non-biotinylated INSL3; lane 4, recombinantLGR8 detected using the M1 antibody.

[0014]FIG. 7. Expression of LGR8 transcripts in the gubernaculum andINSL3 stimulation of cAMP production and thymidine incorporation bycultured gubernacular cells. A) Northern blot analyses. G: gubernaculum;D: diaphragm; T, testis. B) Stimulation of cAMP production in primarycultures of gubernacular cells treated with rat INSL3, porcine relaxin(RLX) or glucagon (Glu). Some cells were treated with foskolin (FS)served as positive controls whereas diaphragm muscle cells served asnegative controls. C) Stimulation of thymidine incorporation by culturedgubernacular cells treated with different hormones for 24 h.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0015] High affinity relaxin receptors, polypeptide compositions relatedthereto, as well as nucleotide compositions encoding the same, areprovided. The subject polypeptide and/or nucleic acid compositions finduse in a variety of different applications, including the identificationof homologous or related genes; for the identification of endogenousligands for these novel receptors; the production of compositions thatmodulate the expression or function of the receptors; for gene therapy;for mapping functional regions of the receptors; in studying associatedphysiological pathways; for in vivo prophylactic and therapeuticpurposes; as immunogens for producing antibodies; in screening forbiologically active agents; and the like.

[0016] Relaxin is a hormone with a number of important functions, whichinclude the modulation of the reproductive physiology of human beingsand other mammals, including, but not limited to, maintaining pregnancy,effecting parturition, and enhancing sperm motility as an aid infertilization. Relaxin has significant effects on connective tissue, asevidenced by its role in pregnancy, for example on the pubic symphysisand rearrangement of collagenous filaments effecting parturition;depressant effects on the myometrium; preparation of the endometrium forimplantation; role in luteolysis; growth and differentiation of themammary glands; enhancement of sperm motility; and augmentation of theability of sperm to penetrate the human cervix. Relaxin has beenimplicated in the dilation of cardiac and blood vessels' smooth musclecells, and has also been used in the treatment of severe chronic pain,particularly pain arising from stretching, swelling, or dislocation oftissues.

[0017] Human relaxin and its methods of preparation, including synthesisin recombinant cell culture, are known. Included within the scope of theterm “relaxin” are human relaxins from recombinant or native sources aswell as relaxin variants, such as amino acid sequence variants. Thepredominant species of human relaxin in the corpus luteum and serum isthe H2 relaxin form with a truncated B chain, i.e., relaxin H2(B29 A24),wherein the four C-terminal amino acids of the B-chain are absent. Alsoincluded within the scope of the term “human relaxin” are otherinsertions, substitutions, or deletions of one or more amino acidresidues, glycosylation variants, unglycosylated human relaxin, organicand inorganic salts, covalently modified derivatives of human relaxin,human preprorelaxin, and human prorelaxin.

[0018] Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0019] In this specification and the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Characterization of LGR7 and LRG8

[0020] LGR7 and LRG8 are mammalian high affinity relaxin receptors,which are of the G-protein coupled, seven trans-membrane family ofproteins, specifically the subfamily of G-protein coupled seventrans-membrane proteins that are characterized by the presence ofextra-cellular leucine rich repeat regions. LRG8 is novel; LRG7 isdescribed in co-pending patent application U.S. Ser. No. 09/647,067.These proteins have both a G-protein coupled seven trans-membrane regionand a leucine rich repeat extra-cellular domain. Both of these receptorsmediate the production of cAMP in response to binding of relaxin, whichproduction is inhibited by the addition of anti-relaxin antibodies.

[0021] The human LGR7 gene encodes two splicing variants (see Hsu et al.2000), of 757 and 723 amino acids, respectively. These molecules may bereferred to as LGR7(1) and LGR7(2), respectively, or generically asLGR7. LGR7 is expressed in multiple tissues, including testis, ovary,prostate, intestine and colon. Expression of LGR7 is cell type-specificin different rodent tissues. In the uterus, the expression of LGR7 ismainly in the endometrial and muscularis layers but minimal in stromaland interstitial cells, consistent with the utero-muscular modulatingactivity of relaxin. In the cervix, LGR7 was found in all muscularislayer. In contrast, negligible staining is found in the skeletal muscle.

[0022] The sequence of LGR8 is provided as SEQ ID NO:1, and encodes apolypeptide of 754 amino acids (SEQ ID NO:2). LGR8 is mainly expressedin the brain, kidney, muscle, testis, thyroid, uterus, bone marrow andperipheral blood cells. In addition to relaxin, LGR8 is a receptor forINSL3, an insulin-like protein related to relaxin (Adham et al. (1993)J. Biol. Chem. 268:26668-26672; Burkhardt et al. (1994) Genomics 20:13-19). InsL3, is expressed exclusively in prenatal and postnatal Leydigcells. INSL3 has a role in the development of the male urogenital tractand in female fertility. Mutations in InsL3 are associated withcryptorchidism.

[0023] Soluble fragments of LGR7 and LGR8 are constructed by deletion ofthe transmembrane domain of the receptor. For example, a soluble form ofLGR7 is made by truncating the protein to delete the transmembranedomain at L402. Gain of function mutations in LGR7(1); LGR7(2) and LGR8are made by amino acid substitution, for example in the molecules LGR8D647Y, and LGR7(1) D637Y. These mutated receptors show increased basallevels of activity.

LGR7 and LGR8 Polypeptides

[0024] For use in the subject methods, either of the native LGR7 or LGR8forms, modifications thereof, or a combination of forms may be used.Polypeptides of interest include the complete mature protein, solublefragments derived therefrom, relaxin-binding domains, mutations whichmay be a gain of function or loss of function mutation, and otherderivatives and fragments thereof. A fragment of a LGR7 or LGR8 peptidemay be selected to achieve a specific purpose, including solubility,isolation of specific domains and binding regions, and the like.Fragments will usually comprise at least about 10 amino acids of theprovided amino acid sequences, and comprise 25, 50, 100 or up to thecomplete polypeptide sequence.

[0025] The sequence of the LGR7 or LGR8 polypeptide may be altered invarious ways known in the art to generate targeted changes in sequence.The polypeptide will usually be substantially similar to the sequencesprovided herein, i.e. will differ by at least one amino acid, and maydiffer by at least two but not more than about ten amino acids. Thesequence changes may be substitutions, insertions or deletions. Scanningmutations that systematically introduce alanine, or other residues, maybe used to determine key amino acids. Conservative amino acidsubstitutions typically include substitutions within the followinggroups: (glycine, alanine); (valine, isoleucine, leucine); (asparticacid, glutamic acid); (asparagine, glutamine); (serine, threonine);(lysine, arginine); or (phenylalanine, tyrosine).

[0026] Modifications of interest that do not alter primary sequenceinclude chemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

[0027] Also included in the subject invention are polypeptides that havebeen modified using ordinary molecular biological techniques andsynthetic chemistry so as to improve their resistance to proteolyticdegradation or to optimize solubility properties or to render them moresuitable as a therapeutic agent. For examples, the backbone of thepeptide may be cyclized to enhance stability (see Friedler et al. (2000)J. Biol. Chem. 275:23783-23789). Analogs of such polypeptides includethose containing residues other than naturally occurring L-amino acids,e.g. D-amino acids or non-naturally occurring synthetic amino acids.

[0028] The subject peptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example automated synthesizers by AppliedBiosystems Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

[0029] If desired, various groups may be introduced into the peptideduring synthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

[0030] The polypeptides may also be isolated and purified in accordancewith conventional methods of recombinant synthesis. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. For the most part, the compositionswhich are used will comprise at least 20% by weight of the desiredproduct, more usually at least about 75% by weight, preferably at leastabout 95% by weight, and for therapeutic purposes, usually at leastabout 99.5% by weight, in relation to contaminants related to the methodof preparation of the product and its purification. Usually, thepercentages will be based upon total protein.

Compound Screening

[0031] The availability of purified LGR7 or LGR8 and other components inthe signaling pathways, e.g. relaxin, InsL3, altered copies of thesemolecules, etc., allows in vitro reconstruction of the signalingpathway. Two or more of the components may be combined in vitro, and thebehavior assessed in terms of production of cAMP; modification ofprotein components, e.g. connective tissues; ability of differentprotein components to bind to each other etc. The components may bemodified by sequence deletion, substitution, etc. to determine thefunctional role of specific residues.

[0032] Drug screening may be performed using an in vitro model, agenetically altered cell or animal, or purified LGR7 or LGR8 protein.One can identify ligands or substrates that compete with, modulate ormimic the action of LGR7 or LGR8. Areas of investigation include thedevelopment of treatments for altering connective tissue, which may bein connection with pregnancy and birth, with disease states such asscleroderma and fibromyalgia, with the treatment of pain associated withdistortions in connective tissue; with the treatment of cryptorchidism;the treatment of endometriosis; the relaxation of smooth muscle cells;and the like.

[0033] Drug screening identifies agents that mimic LGR7 or LGR8activity, either as an antagonist or as an agonist. A wide variety ofassays may be used for this purpose, including labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, and the like. Knowledge of the3-dimensional structure of LGR7 or LGR8, derived from crystallization ofpurified synthetic LGR7 or LGR8 protein, leads to the rational design ofsmall drugs that specifically inhibit LGR7 or LGR8 activity.

[0034] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of altering or mimickingthe physiological function of LGR7 or LGR8. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically one of these concentrations serves as a negative control, i.e.at zero concentration or below the level of detection.

[0035] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0036] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0037] Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0038] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The mixture of components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4 and 40° C. Incubation periodsare selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

[0039] Relaxin or analogs thereof may be useful in the screening assays,as competitors, controls, in structural studies of binding sites, etc. Anumber of such molecules have been described, for example, see U.S. Pat.Nos. 6,200,953; 5,326,694; 5,320,953; 5,179,195; 5,145,962; 5,053,488;5,023,321; 4,871,670; 4,758,516; and 4,656,249.

[0040] The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of connective tissue disorders, during pregnancy etc. Thecompounds may be administered in a variety of ways, orally, topically,parenterally e.g. subcutaneously, intraperitoneally, by viral infection,intravascularly, etc. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-10 wt %.

Antibodies Specific for LGR7 or LGR8 Polypeptides

[0041] The present invention provides antibodies specific for LGR7 orLGR8 polypeptides, e.g. any one of the variants, polypeptides, ordomains described above. Such antibodies are useful, for example, inmethods of detecting the presence of LGR7 or LGR8 in a biologicalsample, and in methods of isolating LGR7 or LGR8 from a biologicalsample.

[0042] The LGR7 or LGR8 polypeptides of the invention are useful for theproduction of antibodies, where short fragments provide for antibodiesspecific for the particular polypeptide, and larger fragments or theentire protein allow for the production of antibodies over the surfaceof the polypeptide. As used herein, the term “antibodies” includesantibodies of any isotype, fragments of antibodies which retain specificbinding to antigen, including, but not limited to, Fab, Fv, scFv, and Fdfragments, chimeric antibodies, humanized antibodies, single-chainantibodies, and fusion proteins comprising an antigen-binding portion ofan antibody and a non-antibody protein. The antibodies may be detectablylabeled, e.g., with a radioisotope, an enzyme which generates adetectable product, a green fluorescent protein, and the like. Theantibodies may be further conjugated to other moieties, such as membersof specific binding pairs, e.g., biotin (member of biotin-avidinspecific binding pair), and the like. The antibodies may also be boundto a solid support, including, but not limited to, polystyrene plates orbeads, and the like.

[0043] “Antibody specificity”, in the context of antibody-antigeninteractions, is a term well understood in the art, and indicates that agiven antibody binds to a given antigen, wherein the binding can beinhibited by that antigen or an epitope thereof which is recognized bythe antibody, and does not substantially bind to unrelated antigens.Methods of determining specific antibody binding are well known to thoseskilled in the art, and can be used to determine the specificity ofantibodies of the invention for a LGR7 or LGR8 polypeptide, particularlya human LGR7 or LGR8 polypeptide.

[0044] Antibodies are prepared in accordance with conventional ways,where the expressed polypeptide or protein is used as an immunogen, byitself or conjugated to known immunogenic carriers, e.g. KLH, pre-SHBsAg, other viral or eukaryotic proteins, or the like. Variousadjuvants may be employed, with a series of injections, as appropriate.For monoclonal antibodies, after one or more booster injections, thespleen is isolated, the lymphocytes immortalized by cell fusion, andthen screened for high affinity antibody binding. The immortalizedcells, i.e. hybridomas, producing the desired antibodies may then beexpanded. For further description, see Monoclonal Antibodies: ALaboratory Manual, Harlow and Lane eds., Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1988. If desired, the mRNAencoding the heavy and light chains may be isolated and mutagenized bycloning in E. coli, and the heavy and light chains mixed to furtherenhance the affinity of the antibody. Alternatives to in vivoimmunization as a method of raising antibodies include binding to phagedisplay libraries, usually in conjunction with in vitro affinitymaturation.

Uses of LGR7 or LGR8 Agonists and Antagonists

[0045] As receptors for relaxin and InsL3, LGR7 and LGR8 have importantroles in the physiology of pregnancy, reproductive development, otherbiological processes relating to smooth muscle and to connective tissue;and the like. Formulations of LGR8 or LGR9, particularly the solublereceptor, as well as other agents that act as agonists or antagonists ofthese receptors find clinical use.

[0046] Agonists or other molecules that simulate the effect of relaxinaffect epithelial cells, blood vessels, stromal cells (putativefibroblasts), and smooth muscle in the cervix and vagina, e.g. bypromoting the onset of labor, increasing endometrial cells, inducingsynthesis of mucins, regulating pituitary prolactin, oxytocin, andvasopressin release, etc.

[0047] These molecules also have important effects on the vascularsystem. Agonists, such as relaxin, are angiogenic in the endometriallining, and plays a role in the attachment of the embryo to the uterus.They can be administered to increase blood flow and vasodilation ofvascular beds. Methods for the use of relaxin to increase angiogenesisare described in U.S. Pat. No. 6,211,147. Relaxin and other agonists canact as a factor in protection against arteriosclerosis and ischemic orthrombotic pathologies, by inducing dilation of blood vessels' smoothmuscle cells which results in an increment of blood flow; inhibitscoagulation processes, intensifies the fibrinolysis and lowers bloodconcentration of lipids and sodium. This effect is mediated bothdirectly, and through release NO and ANP, which largely contribute tothe effect on vessel walls and blood components. See, for example, U.S.Pat. No. 5,952,296.

[0048] Agonists also act as an anti-fibrinolytic agent by decreasingcollagen production, increasing collagen breakdown, and reducing theproduction of the collagenase inhibitor, TIMP Agonists may act directlyon stromal cells to promote remodeling of the extracellular matrix.

[0049] Agonist-induced remodeling of connective tissue has potential forclinical applications, for example in the treatment of systemicsclerosis, or scleroderma, and as a cervical softening agent at term.Conversely, antagonists of LGR7 or LGR8 find use in the prevention oflabor, for example to inhibit pre-term labor.

[0050] Agonists of LGR7 or LGR8 also find use in the treatment offibromyalgia, and may also include the treatment of neurologicaldisorders, for example Alzheimer's disease, Parkinson's, and/or otherconditions such as ADD.

[0051] Another use of the LGR7 or LGR8 agonists is as an analgesic andpalliative for intractable pain (see U.S. Pat. No. 5,656,592). Althoughrelaxin and other agonists can be used generally as an analgesic andpalliative for pain, the conditions most amenable to its therapeuticadministration are those in which unusual stress is chronically placedon tissues because of an acquired or inherent malformation which resultsin the displacement of tissues from their natural disposition in thebody. These agents finds utility, for example, in the treatment ofsevere chronic pain, particularly pain arising from stretching,swelling, or dislocation of tissues.

[0052] Agonists of LGR8 in particular find use in the treatment ofcryptorchidism, a condition that is related to the ligand of LGR8,InsL3. The term cryptorchidism indicates a testis, which has failed todescend to the scrotum and is located at any point along the normal pathof descent or at an ectopic site. Hormones play a pivotal role intesticular descent except during the migration to the level of internalinguinal ring. Cryptorchidism is present in about 4.5% of newborns witha higher incidence in preterms. The incidence decreases to 1.2% by thefirst year. It is classified as palpable and impalpable. The most commonsite of an ectopic testis is superficial inguinal pouch. Retractiletestis is often bilateral and most common in boys between 5 and 6 yearsof age. Hypospadias and inguinal hernias are the most common associatedanomalies seen with undescended testis. Common complications includetorsion and atrophy of testis. Infertility is seen in about 40% ofunilateral and 70% of bilateral cryptorchidism. Undescended testis is 20to 40 times more likely to undergo malignant transformation than normaltestis.

Formulations

[0053] The compounds of this invention can be incorporated into avariety of formulations for therapeutic administration. Particularly,agents specifically bind to and activate LGR7 or LGR8; agents that blockbinding of native ligands, e.g. relaxin or InsL3, to LGR7 or LGR8;agents that modulate expression of LGR7 or LGR8; LGR7 or LGR8polypeptides and analogs or fragments thereof; etc., are formulated foradministration to patients for various clinical purposes, as previouslydescribed. More particularly, the compounds of the present invention canbe formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The agents may be systemic after administration or maybe localized by the use of an implant that acts to retain the activedose at the site of implantation.

[0054] In pharmaceutical dosage forms, the compounds may be administeredin the form of their pharmaceutically acceptable salts, or they may alsobe used alone or in appropriate association, as well as in combinationwith other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0055] For oral preparations, the compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0056] The compounds can be formulated into preparations for injectionsby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

[0057] The compounds can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0058] Furthermore, the compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

[0059] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore compounds of the present invention. Similarly, unit dosage formsfor injection or intravenous administration may comprise the compound ofthe present invention in a composition as a solution in sterile water,normal saline or another pharmaceutically acceptable carrier.

[0060] Implants for sustained release formulations are well-known in theart. Implants are formulated as microspheres, slabs, etc. withbiodegradable or non-biodegradable polymers. For example, polymers oflactic acid and/or glycolic acid form an erodible polymer that iswell-tolerated by the host. The implant is placed in proximity to thetargeted site, so that the local concentration of active agent isincreased relative to the rest of the body.

[0061] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0062] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0063] Typical dosages for systemic administration range from 0.1 μg to100 milligrams per kg weight of subject per administration. A typicaldosage may be one tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient. The time-releaseeffect may be obtained by capsule materials that dissolve at differentpH values, by capsules that release slowly by osmotic pressure, or byany other known means of controlled release.

[0064] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

[0065] The use of liposomes as a delivery vehicle is one method ofinterest. The liposomes fuse with the cells of the target site anddeliver the contents of the lumen intracellularly. The liposomes aremaintained in contact with the cells for sufficient time for fusion,using various means to maintain contact, such as isolation, bindingagents, and the like. In one aspect of the invention, liposomes aredesigned to be aerosolized for pulmonary administration. Liposomes maybe prepared with purified proteins or peptides that mediate fusion ofmembranes, such as Sendai virus or influenza virus, etc. The lipids maybe any useful combination of known liposome forming lipids, includingcationic lipids, such as phosphatidylcholine. The remaining lipid willnormally be neutral lipids, such as cholesterol, phosphatidyl serine,phosphatidyl glycerol, and the like.

LGR7 or LGR8 Nucleic Acids

[0066] The invention includes novel nucleic acids having a sequence setforth in SEQ ID NO:1; nucleic acids that hybridize under stringentconditions, particularly conditions of high stringency, to the sequenceset forth in SEQ ID NO:1; genes corresponding to the provided nucleicacids; sequences encoding the polypeptide set forth in SEQ ID NO:2(LGR8); and fragments and derivatives thereof.

[0067] The nucleic acids of the invention include nucleic acids havingsequence similarity or sequence identity to SEQ ID NO:1. Nucleic acidshaving sequence similarity are detected by hybridization under lowstringency conditions, for example, at 50° C. and 10×SSC (0.9 Msaline/0.09 M sodium citrate) and remain bound when subjected to washingat 55° C. in 1×SSC. Sequence identity can be determined by hybridizationunder stringent conditions, for example, at 50° C. or higher and 0.1×SSC(9 mM saline/0.9 mM sodium citrate). Hybridization methods andconditions are well known in the art, see, e.g., U.S. Pat. No.5,707,829. Nucleic acids that are substantially identical to theprovided nucleic acid sequence, e.g. allelic variants, geneticallyaltered versions of the gene, etc., bind to SEQ ID NO:1 under stringenthybridization conditions. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes. Thesource of homologous genes can be any species, e.g. primate species,particularly human; rodents, such as rats and mice; canines, felines,bovines, ovines, equines, fish, yeast, nematodes, etc.

[0068] In one embodiment, hybridization is performed using at least 18contiguous nucleotides (nt) of SEQ ID NO:1 or a DNA encoding thepolypeptide of SEQ ID NO:2. Such a probe will preferentially hybridizewith a nucleic acid comprising the complementary sequence, allowing theidentification and retrieval of the nucleic acids that uniquelyhybridize to the selected probe. Probes of more than 18 nt can be used,e.g., probes of from about 18 nt to about 25, 50, 100, 250, or 500 nt,but 18 nt usually represents sufficient sequence for uniqueidentification.

[0069] Nucleic acids of the invention also include naturally occurringand synthetically produced variants of the nucleotide sequences (e.g.,degenerate variants, gain of function mutations, soluble forms, allelicvariants, etc.). Variants of the nucleic acids of the invention areidentified by hybridization of putative variants with nucleotidesequences disclosed herein, preferably by hybridization under stringentconditions. For example, by using appropriate wash conditions, variantsof the nucleic acids of the invention can be identified where theallelic variant exhibits at most about 25-30% base pair (bp) mismatchesrelative to the selected nucleic acid probe. In general, allelicvariants contain 15-25% bp mismatches, and can contain as little as even5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.

[0070] The invention also encompasses homologs corresponding to thenucleic acids of SEQ ID NO:1 or a DNA encoding the polypeptide of SEQ IDNO:2, where the source of homologous genes can be any mammalian species,e.g., primate species, particularly human; rodents, such as rats;canines, felines, bovines, ovines, equines, fish, yeast, nematodes, etc.Between mammalian species, e.g., human and mouse, homologs generallyhave substantial sequence similarity, e.g., at least 75% sequenceidentity, usually at least 90%, more usually at least 95% betweennucleotide sequences. Sequence similarity is calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 contiguous nt long, moreusually at least about 30 nt long, and may extend to the completesequence that is being compared. Algorithms for sequence analysis areknown in the art, such as gapped BLAST, described in Altschul, et al.Nucleic Acids Res. (1997) 25:3389-3402.

[0071] The subject nucleic acids can be cDNAs or genomic DNAs, as wellas fragments thereof, particularly fragments that encode a biologicallyactive polypeptide and/or are useful in the methods disclosed herein(e.g., in diagnosis, as a unique identifier of a differentiallyexpressed gene of interest, etc.) The term “cDNA” as used herein isintended to include all nucleic acids that share the arrangement ofsequence elements found in native mature mRNA species, where sequenceelements are exons and 3′ and 5′ non-coding regions. Normally mRNAspecies have contiguous exons, with the intervening introns, whenpresent, being removed by nuclear RNA splicing, to create a continuousopen reading frame encoding a polypeptide of the invention.

[0072] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It can further include the 3′ and 5′untranslated regions found in the mature mRNA. It can further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA can be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ and 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue, stage-specific, or disease-state specificexpression.

[0073] The nucleic acid compositions of the subject invention can encodeall or a part of the subject polypeptides. Double or single strandedfragments can be obtained from the DNA sequence by chemicallysynthesizing oligonucleotides in accordance with conventional methods,by restriction enzyme digestion, by PCR amplification, etc. Isolatednucleic acids and nucleic acid fragments of the invention comprise atleast about 18, about 50, about 100, to about 500 contiguous nt selectedfrom the nucleic acid sequence as shown in SEQ ID NO:1. For the mostpart, fragments will be of at least 18 nt, usually at least 25 nt, andup to at least about 50 contiguous nt in length or more.

[0074] Probes specific to the nucleic acid of the invention can begenerated using the nucleic acid sequence disclosed in SEQ ID NO:1 or aDNA encoding the polypeptide of SEQ ID NO:2. The probes are preferablyat least about 18 nt, 25nt or more of the corresponding contiguoussequence. The probes can be synthesized chemically or can be generatedfrom longer nucleic acids using restriction enzymes. The probes can belabeled, for example, with a radioactive, biotinylated, or fluorescenttag. Preferably, probes are designed based upon an identifying sequenceof one of the provided sequences. More preferably, probes are designedbased on a contiguous sequence of one of the subject nucleic acids thatremain unmasked following application of a masking program for maskinglow complexity (e.g., BLASTX) to the sequence., i.e., one would selectan unmasked region, as indicated by the nucleic acids outside the poly-nstretches of the masked sequence produced by the masking program.

[0075] The nucleic acids of the subject invention are isolated andobtained in substantial purity, generally as other than an intactchromosome. Usually, the nucleic acids, either as DNA or RNA, will beobtained substantially free of other naturally-occurring nucleic acidsequences, generally being at least about 50%, usually at least about90% pure and are typically “recombinant,” e.g., flanked by one or morenucleotides with which it is not normally associated on a naturallyoccurring chromosome.

[0076] The nucleic acids of the invention can be provided as a linearmolecule or within a circular molecule, and can be provided withinautonomously replicating molecules (vectors) or within molecules withoutreplication sequences. Expression of the nucleic acids can be regulatedby their own or by other regulatory sequences known in the art. Thenucleic acids of the invention can be introduced into suitable hostcells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

Modulation of LGR7 or LGR8 Activity

[0077] The LGR7 or LGR8 genes, gene fragments, or the encoded protein orprotein fragments are useful in gene therapy to treat conditionsassociated with LGR7 or LGR8 activity. Inhibition is achieved in anumber of ways. Antisense or siRNA LGR7 or LGR8 sequences may beadministered to inhibit expression. Competitive binding antagonists, forexample, a polypeptide that mimics LGR7 or LGR8 binding may be used toinhibit activity. Other inhibitors are identified by screening forbiological activity in an LGR7 or LGR8 based binding assay. Upregulatingactivity is also of interest, for example through the introduction ofmutations have a gain of function mutation, through increasingexpression levels, and through administering agents that bind to andactivate LGR7 or LGR8.

[0078] Expression vectors may be used to introduce the LGR7 or LGR8 geneinto a cell. Such vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

[0079] The gene or LGR7 or LGR8 peptide may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992)Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992) Nature 356:152-154), where gold microprojectiles arecoated with the LGR7 or LGR8 or DNA, then bombarded into skin cells.

[0080] Antisense molecules can be used to down-regulate expression ofLGR7 or LGR8 in cells. The anti-sense reagent may be antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such anti-sense molecules as RNA. The antisense sequence iscomplementary to the mRNA of the targeted gene, and inhibits expressionof the targeted gene products. Antisense molecules inhibit geneexpression through various mechanisms, e.g. by reducing the amount ofmRNA available for translation, through activation of RNAse H, or sterichindrance. One or a combination of such molecules may be administered,where a combination may comprise multiple different sequences.

[0081] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14:840-844).

[0082] In addition to antisense, small interfering RNA (siRNA) duplexescan be used to inhibit expression of jeb genes. siRNA are doublestranded RNA molecules of at least about 18 nucleotides, and may be upto the length of the complete mRNA. Preferred siRNA for use in mammaliancells are from about 18 to 30 nucleotides, preferably from about 21 to22 nucleotides in length. For example, see Elbashir et al. (2001) Nature411:494-498.

[0083] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993) supra. and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars, heterocyclic bases, morpholinoderivatives, and the like.

[0084] Agents that block LGR7 or LGR8 activity provide a point ofintervention in an important signaling pathway. Numerous agents areuseful in reducing LGR7 or LGR8 activity, including agents that directlymodulate LGR7 or LGR8 expression as described above, e.g. expressionvectors, anti-sense specific for LGR7 or LGR8; and agents that act onthe LGR7 or LGR8 protein, e.g. LGR7 or LGR8 specific antibodies andanalogs thereof, small organic molecules that block LGR7 or LGR8 bindingactivity, etc.

Diagnostic Uses

[0085] Polynucleotide-based reagents derived from the sequence of LGR7or LGR8, e.g. PCR primers, oligonucleotide or cDNA probes, as well asantibodies against LGR7 or LGR8s, are used to screen patient samples,e.g. biopsy-derived tissues, amniotic fluid samples, blood samples,etc., for increased expression of LGR7 or LGR8 mRNA or proteins.DNA-based reagents are also designed for evaluation of chromosomal lociimplicated in certain diseases e.g. for use in loss-of-heterozygosity(LOH) studies, or design of primers based on LGR7 or LGR8 codingsequence. Of particular interest is the use of LGR8 for genetic studiesrelated to the diagnosis of cryptorchidism, which is associated with theexpression of InsL3, and it's activation of LGR8.

[0086] The polynucleotides of the invention can be used to detectdifferences in expression levels between two samples. A differencebetween the protein levels, or the mRNA in the two tissues which arecompared, for example, in molecular weight, amino acid or nucleotidesequence, or relative abundance, indicates a change in the gene, or agene which regulates it, in the tissue or cell sample.

[0087] The subject nucleic acid and/or polypeptide compositions may beused to analyze a patient sample for the presence of polymorphismsassociated with a disease state or genetic predisposition to a diseasestate. Biochemical studies may be performed to determine whether asequence polymorphism in an LGR7 or LGR8 coding region or controlregions is associated with disease. Disease associated polymorphisms mayinclude deletion or truncation of the gene, mutations that alterexpression level, that affect the binding activity of the protein, the Gprotein activity, etc.

[0088] Changes in the promoter or enhancer sequence that may affectexpression levels of LGR7 or LGR8 can be compared to expression levelsof the normal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

[0089] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. a disease associatedpolymorphism. Where large amounts of DNA are available, genomic DNA isused directly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express LGR7 or LGR8 may be used as a source of mRNA, which may beassayed directly or reverse transcribed into cDNA for analysis. Thenucleic acid may be amplified by conventional techniques, such as thepolymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described in Saikiet al. (1985) Science 239:487, and a review of techniques may be foundin Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press1989, pp.14.2-14.33.

[0090] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein(JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

[0091] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to a wild-type LGR7 or LGR8 sequence. Hybridization withthe variant sequence may also be used to determine its presence, bySouthern blots, dot blots, etc. The hybridization pattern of a controland variant sequence to an array of oligonucleotide probes immobilizedon an array, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis(DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonucleases, the sampleis digested with that endonucleases, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

[0092] Screening for mutations in LGR7 or LGR8s may be based on thefunctional or antigenic characteristics of the protein. Proteintruncation assays are useful in detecting deletions that may affect thebiological activity of the protein. Various immunoassays designed todetect polymorphisms in LGR7 or LGR8 proteins may be used in screening.Where many diverse genetic mutations lead to a particular diseasephenotype, functional protein assays have proven to be effectivescreening tools. The activity of the encoded LGR7 or LGR8 protein inbinding assays, etc., may be determined by comparison with the wild-typeprotein. Proteins may also be screened for the presence ofpost-translational modification of the LGR7 or LGR8 proteins, e.g. underpathological conditions, including proteolytic fragments, amidation,acetylation etc.

[0093] Antibodies specific for LGR7 or LGR8 may be used in staining orin immunoassays. Samples, as used herein, include biological fluids suchas blood, amniotic fluid, and the like; organ or tissue culture derivedfluids; and fluids extracted from physiological tissues. Also includedin the term are derivatives and fractions of such fluids. The cells maybe dissociated, in the case of solid tissues, or tissue sections may beanalyzed. Alternatively a lysate of the cells may be prepared.

[0094] Diagnosis may be performed by a number of methods to determinethe absence or presence or altered amounts of normal or abnormal LGR7 orLGR8 in patient cells. For example, detection may utilize staining ofcells or histological sections, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

[0095] In some embodiments, the methods are adapted for use in vivo. Inthese embodiments, a detectably-labeled moiety, e.g., an antibody, whichis specific for LGR7 or LGR8 is administered to an individual (e.g., byinjection), and labeled cells are located using standard imagingtechniques, including, but not limited to, magnetic resonance imaging,computed tomography scanning, and the like.

[0096] Diagnostic screening may also be performed for polymorphisms thatare genetically linked to a disease predisposition, particularly throughthe use of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression. Microsatellite linkage analysis maybe performed alone, or in combination with direct detection ofpolymorphisms, as described above. The use of microsatellite markers forgenotyping is well documented. For examples, see Mansfield et al. (1994)Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib etal., supra.

[0097] The detection methods can be provided as part of a kit. Thus, theinvention further provides kits for detecting the presence of an mRNAencoding LGR7 or LGR8, and/or a polypeptide encoded thereby, in abiological sample. Procedures using these kits can be performed byclinical laboratories, experimental laboratories, medical practitioners,or private individuals. The kits of the invention for detecting apolypeptide comprise a moiety that specifically binds the polypeptide,which may be a specific antibody. The kits of the invention fordetecting a nucleic acid comprise a moiety that specifically hybridizesto such a nucleic acid. The kit may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, standards, instructions, and interpretiveinformation.

Genetically Altered Cell or Animal Models for LGR7 or LGR8 Function

[0098] The subject nucleic acids can be used to generate transgenicanimals or site specific gene modifications in cell lines. Transgenicanimals may be made through homologous recombination, where the normalLGR7 or LGR8 locus is altered. Alternatively, a nucleic acid constructis randomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike.

[0099] The modified cells or animals are useful in the study of LGR7 orLGR8 function and regulation. For example, a series of small deletionsand/or substitutions may be made in the LGR7 or LGR8 gene to determinethe role of different residues in ligand binding, signal transduction,etc. Of interest are the use of LGR7 or LGR8 to construct transgenicanimal models for pregnancy related disorders, connective tissuedisorders, etc. where expression of LGR7 or LGR8 is specifically reducedor absent. Specific constructs of interest include anti-sense LGR7 orLGR8, which will block LGR7 or LGR8 expression and expression ofdominant negative LGR7 or LGR8 mutations. A detectable marker, such aslac Z may be introduced into the LGR7 or LGR8 locus, where up-regulationof LGR7 or LGR8 expression will result in an easily detected change inphenotype.

[0100] One may also provide for expression of the LGR7 or LGR8 gene orvariants thereof in cells or tissues where it is not normally expressedor at abnormal times of development. By providing expression of LGR7 orLGR8 protein in cells in which it is not normally produced, one caninduce changes in cell behavior, e.g. in the control of endometriosis,alterations in connective tissue, and the like.

[0101] DNA constructs for homologous recombination will comprise atleast a portion of the LGR7 or LGR8 gene with the desired geneticmodification, and will include regions of homology to the target locus.The regions of homology may include coding regions, or may utilizeintron and/or genomic sequence. DNA constructs for random integrationneed not include regions of homology to mediate recombination.Conveniently, markers for positive and negative selection are included.Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the art. For various techniquesfor transfecting mammalian cells, see Keown et al. (1990) Methods inEnzymology 185:527-537.

[0102] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

[0103] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on pregnancy and birth, etc.

Experimental

[0104] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLE 1

[0105] Four orphan receptors, LGR4-7, have recently been isolated. Thesehave structural features similar to those of the gonadotropin andthyrotropin receptors. Based on structural motifs and phylogeneticanalysis, the orphan LGRs could be subdivided into two subgroups, withLGR4-6 as one group and LGR7 as another. Orthologous genes for eachsubgroup of LGR have been found in invertebrates. Similar to its snailand fly orthologs, human LGR7 has a unique N-terminal cysteine-rich LDLreceptor-like domain preceding the multiple leucine-rich repeats foundin the ectodomain of all other LGRs. Furthermore, constitutively activeLGR7 mutants showing ligand-independent cAMP production were constructedbased on gain-of-function point mutations found in the LH receptor ofpatients with male-limited precocious puberty.

[0106] A new human LGR is identified herein, which belongs to the samesubgroup as LGR7 and is named LGR8 based on the chronological order ofdiscovery. As shown in FIG. 1A, sequence analysis indicated LGR8 sharedabout 60% identity with LGR7 in both the ectodomain and thetransmembrane region. Phylogenetic analysis showed that LGR8 has theclosest relatedness with LGR7 and a Drosophila orthologous receptor ascompared to other human LGRs (FIG. 1B). Similar to the gain-of-functionmutants identified for LGR7, a LGR8 mutant with a D578G substitution inthe transmembrane VI of this receptor conferred ligand-independentincreases in basal cAMP production in transfected cells (FIG. 1C). Thesefindings suggested that LGR8, like LGR7, could couple with the Gsprotein to activate adenylate cyclase.

[0107] Although both LGR7 and LGR8 are orphan receptors, the similarcryptorchidism phenotypes of InsL3 null mice and mutant mice with adisruption of the mouse GREAT gene (an LGR8 ortholog) led to thehypothesis that the relaxin family of peptide proteins are the ligandsfor LGR7 and LGR8. This hypothesis was reinforced by earlier reportsshowing relaxin stimulation of cAMP production in endometrial, anteriorpituitary and other cells.

[0108] 293T cells expressing human LGR7 or LGR8 were treated withporcine relaxin. As shown in FIGS. 2A and B, relaxin treatment resultedin dose-dependent increases in cAMP production with an EC₅₀ of 0.1 and0.5 nM for LGR7 and LGR8, respectively. In contrast, treatment withstructural homologs, insulin, IGF-I, and IGF-II, as well as an unrelatedpeptide hormone glucagon was ineffective. The present findingsdemonstrate that relaxin is the cognate ligand for two G protein-coupledreceptors, LGR7 and LGR8, capable of activating adenylate cyclases.

[0109] To determine whether the expression of LGR7 and LGR8 isconsistent with previous studies on relaxin target sites, the expressionpattern of LGR7 and LGR8 was determined in diverse human tissues (FIG.3A). Reverse transcription-PCR analysis using a panel of 23 differenthuman cDNAs indicated that LGR7 is expressed in diverse tissues asdemonstrated previously using Northern blotting analysis, whereas LGR8is mainly present in the brain, kidney, muscle, testis, thyroid, uterus,bone marrow and peripheral blood cells. Specific antibodies weregenerated against the ectodomain of LGR7. Immunohistochemical analysisdemonstrated that the expression of LGR7 is cell type-specific indifferent rodent tissues. In the uterus, the expression of LGR7 ismainly in the endometrial and muscularis layers but minimal in stromaland interstitial cells, consistent with the utero-muscular modulatingactivity of relaxin. In the cervix, LGR7 was found in all muscularislayer. In contrast, negligible staining was found in the skeletal muscle(FIG. 3B).

[0110] Studies on the classical LGRs have demonstrated that theectodomains of gonadotropin and thyrotropin receptors are theligand-binding regions and an anchored receptor approach was previouslyused to derive the soluble ectodomains of these receptors as functionalantagonists. Using this strategy, permanent cell lines expressing theectodomain of LGR7 fused to the single transmembrane region of CD8through a thrombin cleavage site were isolated (FIG. 4A). Followingthrombin treatment, mg quantities of the soluble ectodomain of LGR7,named as 7BP, was generated. As shown in FIG. 4B, cross-linking analysisdemonstrated a concentration-dependent formation of complexes betweenrelaxin and 7BP whereas a homologous soluble ectodomain from rat LGR4(4BP) showed negligible interaction with relaxin. In addition,co-treatment with 7BP dose-dependently blocked the stimulatory effectsof relaxin to activate LGR7 and LGR8 expressed in 293T cells (FIG. 4C),but co-treatment with 4BP was ineffective. Consistent with an earlierfinding, treatment with relaxin stimulated cAMP production by culturedrat myometrial cells (FIG. 4D). This stimulatory effect of relaxin wasalso antagonized by co-treatment with 7BP.

[0111] Furthermore, subcutaneous administration of 7BP (500 μg/day) for4 days between post-conception days 17 and 20 in pregnant mice led toparturition delay and nipple malformation (Table 1). TABLE 1 Delay ofParturition and Inhibition of Nipple Development by 7BP Control 7BPtreatment Pregnancy Duration 508.8 ± 8.9 532.2 ± 9.0  (hours) Nipplesize  1.55 ± 0.1 1.01 ± 0.06 (length × width, mm²)

[0112] Prolonged duration of straining was found. There were lowerincidences of normal maternal behavior observed at birth. In addition,little or no milk was observed in the abdomen of most live pups of7BP-treated mice, whereas abundant milk was observed in the abdomen ofall live pups of control mice. The inability of 7BP-treated mother tonurse the young is consistent with findings in relaxin null mice.Although earlier studies have used neutralizing antibodies to relaxin todelay parturition in pregnant rats, a more pronounced antagonisticeffect of 7BP was observed here, suggesting that other endogenousligands for LGR7, in addition to relaxin, could be important in theparturition process.

[0113] The present identification of two orphan G protein-coupledreceptors as relaxin receptors is in direct contrast to the well-knownsignaling mechanism of insulin and IGFs mediated by the tetramerictyrosine kinase receptors. Although relaxin has been traditionallyclassified in the insulin family of hormones based on similar domainarrangements, the present results indicated that relaxin and relatedligands could have diverged from insulin/IGF ligands before separationof the arthropods because an ortholog of LGR7 and LGR8 could be found inthe fly genome (FIG. 1C) in addition to receptors homologous to theother two subgroups of mammalian LGRs (9).

[0114] The insulin ligand-signaling receptor system has been found to beimportant in nutrition, longevity, and reproduction in the C. elegans.It is likely that a subgroup of relaxin-related ligands evolved early tosubserve tissue remodeling functions including actions on reproductivetracts in modern mammals. Indeed, a relaxin-like gene has been found inthe primitive tunicates whereas a putative insulin-like protein inDrosophila exhibited greater sequence homology to mammalian relaxin thanto insulin and IGFs. The proposed evolution of divergent receptormechanisms for relaxin and insulin are consistent with their crystalstructure analyses. Although relaxin, like insulin, crystallizes as adimer, the orientation of the molecules in the respective dimers iscompletely different. Because the dimer interface determinants proposedfor receptor binding for insulin and relaxin are quite different, it wasproposed that these two structurally related hormones have evolvedsomewhat dissimilar mechanisms for receptor binding.

[0115] The identification of cAMP as a second messenger for relaxin isconsistent with earlier findings of relaxin stimulation of cAMPproduction by human endometrial and rat pituitary cells as well as mousepubic symphysis and rat cervical fragments. Unlike insulin, relaxin genesequences are highly variable among different vertebrate speciesstudied. The present availability of recombinant human LGR7 and LGR8 canprovide uniform and convenient in vitro bioassays for relaxin, whereasthe derivation of the soluble ligand-binding ectodomains can serve asfunctional antagonists for relaxin during tissue remodeling processesand pregnancy in human and lower species.

[0116] Although relaxin activates both LGR7 and LGR8 based on studies ofrecombinant receptors (FIG. 2), LGR7 is likely to mediate both endocrineand paracrine actions of relaxin whereas LGR8 is more important for aparacrine role, based on the observed lower sensitivity of LGR8 torelaxin. The existence of two relaxin receptors with overlapping tissueexpression patterns raises issues regarding their respectivephysiological roles. LGR7 is expressed in tissues known to possessrelaxin binding sites, including ovary, testis, uterus, brain, andheart. Because LGR7 transcript was also found in many tissues notpreviously known to be relaxin targets, it is interesting to analyze thepotential action of relaxin in these tissues. In contrast, theexpression of LGR8 is more restricted.

[0117] Although antepartum increases in circulating relaxin has beenfound in rat and selected species, relaxin may be a paracrine hormone inhumans and is secreted by corpus luteum, placenta, and uterus tosubserve local functions. The differential expression of LGR7 and LGR8and the possible involvement of additional ligands for these receptorsprovide the basis to elucidate the role of these receptors and theirligands during pregnant states. Identification of LGR7 and LGR8 asrelaxin receptors provides the basis to elucidate the differentialligand specificity and tissue distribution of these two proteins for theunderstanding of the diverse actions of relaxin during pregnant andnonpregnant states.

[0118] Preterm labor and delivery remain a major cause of perinatalmorbidity, mortality, and long-term adverse neurodevelopmental outcome,whereas prolonged labor is also associated with major stress for mothersand infants. Although conflicting outcome has been reported for the useof relaxin as a labor-inducing agent, future studies on the signaltransduction mechanism of relaxin receptors will allow the design ofagonistic or antagonistic relaxin analogs for the treatment of disordersof labor onset. Elucidation of the ligand signaling mechanisms ofrelaxin receptors will also lead to a better understanding of the roleof relaxin and related hormones in uterine and mammary growth. Inaddition to actions on reproductive tissues, relaxin has been shown toregulate pituitary prolactin, oxytocin, and vasopressin release,probably by binding to putative receptors in brain and pituitary. Inaddition, relaxin binding sites have been found in mast cells, and ahuman monocytic cell line.

[0119] Relaxin has important effects on the vascular system. It isangiogenic in the endometrial lining and plays a role in the attachmentof the embryo to the uterus, and structural remodeling of the abdomen,joints and tendons to accommodate the growing fetus. In addition,relaxin regulates the circulatory system to ensure adequate blood flowand oxygenation to the growing fetus. It stimulates vasodilation ofvascular beds by activating the endothelin B receptor subtype andinhibiting the vasoconstrictive effects of angiotensin II. Relaxin alsoacts as an anti-fibrinolytic agent by decreasing collagen production,increasing collagen breakdown, and reducing the production of thecollagenase inhibitor, TIMP. Understanding of the vascular activities ofrelaxin mediated by its receptors will allow stimulation of new bloodvessel growth in selective target tissues and ischemic wound sites.

[0120] Studies on this relaxin receptor will facilitate understandingson connective tissue remodeling and allow new treatments of skinconditions such as scleroderma. Although the major biological effect ofrelaxin is to remodel the mammalian reproductive tract and breast inpregnant females to facilitate the birth process and nursing, highlevels of relaxin secreted by the prostate has also been found in humanseminal plasma and might play a role in sperm motility and fertilizationcapacity. The present elucidation of relaxin receptors could facilitatethe understanding of the role of relaxin in males.

[0121] The present study underscored the value of a genomic approach inthe matching of orphan ligand-receptor pairs. Although relaxin hasdomain arrangements similar to those of the insulin/lGF family ofproteins known to activate tetrameric tyrosine kinase receptors, thecompletely sequenced human genome has only one orphan insulinreceptor-like gene that is unlikely to be the receptor for the divergentrelaxin-like factors. Likewise, the analysis of paralogs of glycoproteinhormone subunit genes indicated that the limited number of remainingcandidate ligands is unlikely to interact with orphan LGRs. Thus, theorphan LGRs are likely to interact with ligands other than theheterodimeric gonadotropins and thyrotropin.

[0122] The present demonstration of LGR7 and LGR8 as relaxin receptorsindicated that a separate ligand signaling system has evolved for therelaxin subfamily of insulin-like genes, including InsL3 and relaxin.Similar to the ancient origin of the insulin ligand/receptor system,structural orthologs forLGR7 and LGR8 could be traced to fly and snail,and phylogenetic analysis indicated that this subgroup of LGRs evolvedbefore the emergence of Bilateria. Thus, the usage of two distinctreceptor-signaling mechanisms for structurally-related insulin andrelaxin family of peptide ligands is likely to be ancient in origin. Itis becoming clear that the actions of relaxin and its related ligands(e.g. InsL3) are mediated by at least two related LGRs (7 and 8). Basedon the hypothesized coevolution of the relaxin family of ligands and theorphan LGRs, future studies on the matching of InsL3 and relatedrelaxin-like ligands (InsL4, RIF1, and RIF2) with LGR8 and the remainingorphan LGRs (LGR4-6) are of interest.

EXAMPLE 2

[0123] INSL3, also known as Leydig insulin-like peptide or relaxin-likefactor, is a relaxin family member expressed in testis Leydig cells andovarian theca and luteal cells. Male mice mutant for INSL3 exhibitcryptorchidism or defects in testis descent due to abnormal gubernaculumdevelopment whereas overexpression of INSL3 induces ovary descent intransgenic females. Because transgenic mice missing the LGR8 gene arealso cryptorchid, INSL3 was tested as the ligand for LGR8. Here, it isshown that treatment with INSL3 stimulated cAMP production in cellsexpressing recombinant LGR8, but not LGR7. In addition, interactionsbetween INSL3 and LGR8 were demonstrated following ligand receptorcross-linking. Northern blot analysis indicated that the LGR8transcripts are expressed in gubernaculum whereas treatment of culturedgubernacular cells with INSL3 stimulated cAMP production and thymidineincorporation. Demonstration of INSL3 as the ligand for LGR8 facilitatesunderstanding of the mechanism of testis descent and allows studies onthe role of INSL3 in gonadal and other physiological processes.

[0124] Experimental Procedures:

[0125] Ovine and rat INSL3 were chemically synthesized and characterizedas previously described. Human INSL3 and biotinylated ovine INSL3 wereprepared similarly with the ovine INSL3 containing a single biotinmolecule on the N-terminus of the A chain. The National Hormone andPituitary Program (NIDDK, National Institutes of Health, Bethesda, Md.)supplied porcine relaxin. ¹²⁵I-Streptavidin and streptavidin conjugatedto horseradish peroxidase (HRP) were purchased from AmershamBiosciences, Inc (Piscataway, N.J.), whereas foskolin, glucagon,collagenase and trypsin were from Sigma Chemical Co. (St. Louis, Mo.).Sprague-Dawley rats were obtained from Simonsen Laboratories (Gilroy,Calif.). Animals were anesthetized and killed using CO₂. Animal care wasconsistent with institutional and NIH guidelines.

[0126] Human 293T cells were maintained in Dulbecco's modified Eagle'smedium/Ham's F-12 (DMEM/F12) supplemented with 10% fetal bovine serum(FBS), 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mML-glutamine. When 70-80% confluent, cells were transfected with 10 μg ofplasmid using the calcium phosphate precipitation method. After 18-24 hof incubation, media were replaced with DMEM/F12 containing 10% FBS.Forty-eight hours after transfection, cells (10⁵/ml) were preincubatedat 37° C. for 30 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma Chemical Co.) before treatment with or withouthormones for 12 h. Total cAMP content was measured in triplicate by aspecific radioimmunoassay. All experiments were repeated at least fourtimes using cells from independent transfections.

[0127] To estimate INSL3 binding, transfected cells were washed twicewith D-PBS and collected in D-PBS before centrifugation at 400×g for 5min. Cells pellets were resuspended in D-PBS containing 1 mg/ml BSA andincubated with increasing doses of the rat INSL3 at 4° C. for 24 h inthe presence of biotinylated INSL3 (5 nM/tube). After incubation, cellswere centrifuged and washed twice with 1% BSA/PBS before incubation with¹²⁵I-Streptavidin (400,000 cpm/tube) for 1 h at 4° C. After washing thecells three times, radioactivity in the pellets was determined. Forprotein blotting, transfected cells were incubated with biotinylatedINSL3 (50 nM/tube) with or without an excess of rat INSL3 (1 μM/tube).After washing, pellets were incubated in D-PBS with disuccinimidylsuberate (0.5 mM) for 30 min. at room temperature. The cross-linkedINSL3-LGR8 complexes were solubilized with 100 μl 1% Triton X-100 in 50mM Tris-HCl. The lysates were denatured with SDS and2-beta-mercaptoethanol, and fractionated using SDS-PAGE. After blottingonto nitrocellulose membranes (Hybond-P, Amersham) and blocking with a5% milk solution, the blots were incubated for 2 h at room temperaturewith streptavidin (1:10,000 dilution) before development using enhancedchemiluminescence solution (ECL, Amersham Life Science). In addition,epitope-tagged LGR8 was extracted with 1% Triton X-100 from cellstransfected with the LGR8 expression plasmid and incubated with the M1antibody for 1 h. Protein G-Sepharose was subsequently added toprecipitate the M1-tagged receptor protein. The precipitate was furtherfractionated using SDS-PAGE followed by immunoblotting using the M1antibody.

[0128] Total RNA from different rat tissues were extracted using theRNeasy purification kits (QIAGEN Inc. Chatsworth, Calif.) beforeNorthern blotting. Rat orthologs for LGR7 and LGR8 were identified inthe GenBank (accession number AC098607 and AC098990, respectively).These sequences were used in reverse transcription-PCR to yield LGR8 andLGR7 probes of 230 and 226 bp, respectively.

[0129] Gubernacular cells were isolated by modifying an earlier method.Tissues were removed from one-week-old rats and cut into 1 mm pieces,and dissociated for 2 h at 37° C. in DMEM/F12 with 0.1% collagenase.Cell debris was removed by passage through a sterile filter and cellswere collected by centrifugation. After suspension in DMEM/F12 with 10%FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine,cells were cultured for 24 h in 5% CO₂ incubator at 37° C. The cellswere then washed once with serum-free medium and treated in DMEM/F12containing IBMX with or without hormones and reagents. After 16 h ofincubation, total cAMP was measured in triplicates as described above.For thymidine incorporation studies, gubernacular cells (2×10⁵ cells/500μl) were cultured in 5 ml polypropylene Falcon tubes (Becton Dickinson,Franklin Lakes, N.J.) with or without hormones together with 1 μCi/tubeof [methyl-³H]thymidine (Amersham Pharmacia Biotech). After 24 h ofculture, cells were washed once and resuspended with ice-cold PBS beforecentrifugation at 2000×g for 30 min. at 4° C. Radioactivities in thewashed cell samples were determined using a β-photomultiplier.

[0130] Results

[0131] INSL3 is the cognate ligand for LGR8. Although INSL3 binds togubernacular homogenates, and induces growth of rat gubernaculum inorgan cultures, the exact nature of the INSL3 receptor is unknown. Humanfetal kidney 293T cells were transfected with expression vectorsencoding human LGR8 or the related LGR7 for testing of INSL3 signaling.In cells expressing LGR8 (FIG. 5A), treatment with synthetic human,ovine, or rat INSL3 led to dose-dependent increases in cAMP production.Although treatment with biotinylated-ovine INSL3 or porcine relaxin(RLX) was also effective, treatment with glucagon did not increase cAMPproduction. In contrast, cells expressing LGR7 responded only to relaxintreatment whereas treatments with INSL3 from different species or humanglucagon were ineffective (FIG. 5B). These results indicated that INSL3is a specific ligand for LGR8.

[0132] To demonstrate the direct binding of INSL3 to LGR8, cellsexpressing LGR8 were incubated with biotinylated INSL3 with or withoutincreasing doses of non-biotinylated INSL3. Following incubation at 4 C.for 24 h, cells were washed and incubated further with I¹²⁵-labeledstreptavidin to estimate the levels of cell-bound biotinylated INSL3. Asshown in FIG. 6A, specific binding of biotinylated INSL3 to LGR8 couldbe competed by non-biotinylated INSL3 in a dose-dependent manner with anED₅₀ of 12 nM (filled circles). In contrast, 293T cells without LGR8expression did not exhibit specific binding (open triangles). Theformation of the LGR8-INSL3 complexes was further estimated followingcross-linking and protein blotting before signal detection usingavidin-horseradish peroxidase (HRP). As shown in FIG. 6B, biotinylatedINSL3 cross-linked with LGR8 could be detected as a high MW band (˜84KDa) whereas a 20-fold excess of non-biotinylated INSL3 decreased signalintensity (lanes 2 and 3). In contrast, the free biotinylated INSL3migrated at 6.5 KDa (FIG. 6B, lane 1) and the epitope-tagged LGR8extracted from transfected cells migrated at ˜75 KDa when monitoredusing the M1 antibody after immunoprecipitation with the same antibody(FIG. 6B, lane 4).

[0133] Expression of LGR8 in gubernaculum and INSL3 stimulation ofgubernacular functions. Northern blotting analyses demonstrated theexpression of the LGR8 transcript in the gubernaculum of one-week-oldimmature rats and testis of adult rats, but not in diaphragm (FIG. 7A).In the gubernaculum, a single transcript of ˜2.5 kb was evident whereasan additional transcript of a higher size was found in the testis. Inaddition, treatment of gubernacular cells with INSL3 led todose-dependent increases in cAMP production (FIG. 7B) to levelscomparable to cells treated with forskolin (FS), a diterpene adenylcyclase activator. Although glucagon treatment was ineffective,treatment with relaxin also stimulated cAMP production by these cells,consistent with its ability to activate LGR8. For diaphragm cells, noneof the hormones tested elicited cAMP production despite the stimulatoryeffects of forskolin (FIG. 7B). Because an increase in gubernacular celldivision is believed to be needed during testis descent, the ability ofINSL3 to stimulate thymidine incorporation by cultured gubernacularcells was tested. As shown in FIG. 7C, treatment with INSL3 led todose-dependent increases in thymidine incorporation by these cells. Inaddition, treatment with relaxin and forskolin, but not glucagon, wasalso effective.

[0134] The present findings demonstrate that INSL3 is the cognate ligandfor LGR8. The observed expression of LGR8 transcripts in thegubernaculum and the INSL3 stimulation of cAMP production by these cellsare consistent with the common cryptorchid phenotypes of thisligand-receptor pair in earlier transgenic mouse studies. Although thelarge 550 kb DNA deletion induced in transgenic mice following randominsertional mutagenesis includes genes other than the mouse LGR8ortholog, present findings of the ligand-receptor relationship for INLS3and LGR8 supports the hypothesis that deletion of this receptor genealone is responsible for the cryptorchid phenotype. Despite thebilateral cryptorchidism found in male INSL3 null mice as a result ofdevelopmental abnormalities of the gubernaculum, most studies indicatedthat INSL3 gene mutations are not associated with cryptorchidism inpatients. Two putative mutations, R49X and P69L, were identified in theconnecting peptide region of the precursor INSL3 protein. Because thefrequency of these INSL3 gene mutations is low (1.4%), their potentialinfluence on testis descent waits further testing. The presentidentification of LGR8 as the receptor for INSL3 raised the possibilitythat partial or complete loss-of-function mutations in the LGR8 gene areassociated with cryptorchidism, the most frequent congenitalabnormalities in humans.

[0135] INSL3 specifically activates LGR8, but not LGR7. A total of sevenrelaxin members are present in the human genome. Relaxin H1 and H2 areclustered together with INSL4 and INSL6 in chromosome 9p23-24 whereasINSL3 is located together with relaxin 3 in 19p13. The present findingsprovide the basis to test the receptor binding specificity of otherrelaxin paralogs, thus allowing a better understanding of the evolutionand physiology of the relaxin ligand gene family. Based on the divergentreceptor specificity of relaxin and INSL3, future chimeric receptorstudies on the ligand specificity of LGR7 and LGR8 are also of interest.

[0136] During fetal development, the sexual dimorphic position inmammalian gonads is dependent on the differential development of twoligaments. In males, growth of the gubernaculum and regression of thecranial suspensory ligament result in transabdominal descent of thetestes. Circulating INSL3 concentrations increase in male rats startingat day 10 of age and continuing until INSL3 concentrations reached adultlevels at day 39 after parturition. The testicles are descending intothe scrotum during this phase of increasing INSL3 concentrations. INSL3is expressed in Leydig cells of the fetal and postnatal testis and alsoin theca and luteal cells of the postnatal ovary, whereas LGR8 isexpressed in multiple tissues including testis, brain, kidney, muscle,thyroid, uterus, peripheral blood cells, and bone marrow. In addition toits endocrine role in testis descent mediated by LGR8 in gubernaculum,INSL3 could also have important endocrine or paracrine roles in othertissues. Although defective spermatogenesis found in INSL3 or LGR8 nullmice could be the secondary effects of cryptorchidism, Leydigcell-derived INSL3 could play a paracrine role in the testis becauseLGR8 is also expressed in the testis. In females, INSL3 is expressed inthe luteal cells of the ovary through the cycle, and during pregnancy.Because female INSL3 null mice have impaired fertility associated withderegulation of the estrous cycle, the present findings will facilitateunderstanding of the paracrine role of INSL3 in the ovary in addition toproviding understandings on the physiological roles of LGR8 innon-gonadal tissues such as brain, thyroid, and uterus.

[0137] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The publications discussedherein are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such adisclosure by virtue of prior invention.

[0138] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1 4 1 2838 DNA H. sapiens CDS (107)...(2369) 1 actcactata gggctcgagcggccgcccgg gcaggtgaac ttactacatc agaactcctg 60 ctgaggtata agaggatacgtctaataact caattgctgt aaacct atg att gtt 115 Met Ile Val 1 ttt ctg gttttt aaa cat ctc ttc agc ctc aga ttg att aca atg ttc 163 Phe Leu Val PheLys His Leu Phe Ser Leu Arg Leu Ile Thr Met Phe 5 10 15 ttt cta ctt catttc atc gtt ctg atc aat gtc aaa gat ttt gca ctg 211 Phe Leu Leu His PheIle Val Leu Ile Asn Val Lys Asp Phe Ala Leu 20 25 30 35 act caa ggt agcatg atc act cct tca tgc caa aaa gga tat ttt ccc 259 Thr Gln Gly Ser MetIle Thr Pro Ser Cys Gln Lys Gly Tyr Phe Pro 40 45 50 tgt ggg aat ctt accaag tgc tta ccc cga gct ttt cac tgt gat ggc 307 Cys Gly Asn Leu Thr LysCys Leu Pro Arg Ala Phe His Cys Asp Gly 55 60 65 aag gat gac tgt ggg aacggg gcg gac gaa gag aac tgt ggt gac act 355 Lys Asp Asp Cys Gly Asn GlyAla Asp Glu Glu Asn Cys Gly Asp Thr 70 75 80 agt gga tgg gcg acc ata tttggc aca gtg cat gga aat gct aac agc 403 Ser Gly Trp Ala Thr Ile Phe GlyThr Val His Gly Asn Ala Asn Ser 85 90 95 gtg gcc tta aca cag gag tgc tttcta aaa cag tat cca caa tgc tgt 451 Val Ala Leu Thr Gln Glu Cys Phe LeuLys Gln Tyr Pro Gln Cys Cys 100 105 110 115 gac tgc aaa gaa act gaa ttggaa tgt gta aat ggt gac tta aag tct 499 Asp Cys Lys Glu Thr Glu Leu GluCys Val Asn Gly Asp Leu Lys Ser 120 125 130 gtg ccg atg att tct aac aatgtg aca tta ctg tct ctt aag aaa aac 547 Val Pro Met Ile Ser Asn Asn ValThr Leu Leu Ser Leu Lys Lys Asn 135 140 145 aaa atc cac agt ctt cca gataaa gtt ttc atc aaa tac aca aaa ctt 595 Lys Ile His Ser Leu Pro Asp LysVal Phe Ile Lys Tyr Thr Lys Leu 150 155 160 aaa aag ata ttt ctt cag cataat tgc att aga cac ata tcc agg aaa 643 Lys Lys Ile Phe Leu Gln His AsnCys Ile Arg His Ile Ser Arg Lys 165 170 175 gca ttt ttt gga tta tgt aatctg caa ata tta tat ctc aac cac aac 691 Ala Phe Phe Gly Leu Cys Asn LeuGln Ile Leu Tyr Leu Asn His Asn 180 185 190 195 tgc atc aca acc ctc agacct gga ata ttc aaa gac tta cat cag cta 739 Cys Ile Thr Thr Leu Arg ProGly Ile Phe Lys Asp Leu His Gln Leu 200 205 210 act tgg cta att cta gatgac aat cca ata acc aga att tca cag cgc 787 Thr Trp Leu Ile Leu Asp AspAsn Pro Ile Thr Arg Ile Ser Gln Arg 215 220 225 ttg ttt acg gga tta aattcc ttg ttt ttc ctg tct atg gtt aat aac 835 Leu Phe Thr Gly Leu Asn SerLeu Phe Phe Leu Ser Met Val Asn Asn 230 235 240 tac tta gaa gct ctt cccaag cag atg tgt gcc caa atg cct caa ctc 883 Tyr Leu Glu Ala Leu Pro LysGln Met Cys Ala Gln Met Pro Gln Leu 245 250 255 aac tgg gtg gat ttg gaaggc aat aga ata aag tat ctc aca aat tct 931 Asn Trp Val Asp Leu Glu GlyAsn Arg Ile Lys Tyr Leu Thr Asn Ser 260 265 270 275 acg ttt ctg tcg tgcgat tcg ctc aca gtg ctg ttt ctg cct aga aat 979 Thr Phe Leu Ser Cys AspSer Leu Thr Val Leu Phe Leu Pro Arg Asn 280 285 290 caa att ggt ttt gttcca gag aag aca ttt tct tca tta aaa aat tta 1027 Gln Ile Gly Phe Val ProGlu Lys Thr Phe Ser Ser Leu Lys Asn Leu 295 300 305 gga gaa ctg gat ctgtct agc aat acg ata acg gag cta tca cct cac 1075 Gly Glu Leu Asp Leu SerSer Asn Thr Ile Thr Glu Leu Ser Pro His 310 315 320 ctt ttt aaa gac ttgaag ctt cta caa aag ctg aac ctg tca tcc aat 1123 Leu Phe Lys Asp Leu LysLeu Leu Gln Lys Leu Asn Leu Ser Ser Asn 325 330 335 cct ctt atg tat cttcac aag aac cag ttt gaa agt ctt aaa caa ctt 1171 Pro Leu Met Tyr Leu HisLys Asn Gln Phe Glu Ser Leu Lys Gln Leu 340 345 350 355 cag tct cta gacctg gaa agg ata gag att cca aat ata aac aca cga 1219 Gln Ser Leu Asp LeuGlu Arg Ile Glu Ile Pro Asn Ile Asn Thr Arg 360 365 370 atg ttt caa cccatg aag aat ctt tct cac att tat ttc aaa aac ttt 1267 Met Phe Gln Pro MetLys Asn Leu Ser His Ile Tyr Phe Lys Asn Phe 375 380 385 cga tac tgc tcctat gct ccc cat gtc cga ata tgt atg ccc ttg acg 1315 Arg Tyr Cys Ser TyrAla Pro His Val Arg Ile Cys Met Pro Leu Thr 390 395 400 gac ggc att tcttca ttt gag gac ctc ttg gct aac aat atc ctc aga 1363 Asp Gly Ile Ser SerPhe Glu Asp Leu Leu Ala Asn Asn Ile Leu Arg 405 410 415 ata ttt gtc tgggtt ata gct ttc att acc tgc ttt gga aat ctt ttt 1411 Ile Phe Val Trp ValIle Ala Phe Ile Thr Cys Phe Gly Asn Leu Phe 420 425 430 435 gtc att ggcatg aga tct ttc att aaa gct gaa aat aca act cac gct 1459 Val Ile Gly MetArg Ser Phe Ile Lys Ala Glu Asn Thr Thr His Ala 440 445 450 atg tcc atcaaa atc ctt tgt tgt gct gat tgc ctg atg ggt gtt tac 1507 Met Ser Ile LysIle Leu Cys Cys Ala Asp Cys Leu Met Gly Val Tyr 455 460 465 ttg ttc tttgtt ggc att ttc gat ata aaa tac cga ggg cag tat cag 1555 Leu Phe Phe ValGly Ile Phe Asp Ile Lys Tyr Arg Gly Gln Tyr Gln 470 475 480 aag tat gccttg ctg tgg atg gag agc gtg cag tgc cgc ctc atg ggg 1603 Lys Tyr Ala LeuLeu Trp Met Glu Ser Val Gln Cys Arg Leu Met Gly 485 490 495 ttc ctg gccatg ctg tcc acc gaa gtc tct gtt ctg cta ctg acc tac 1651 Phe Leu Ala MetLeu Ser Thr Glu Val Ser Val Leu Leu Leu Thr Tyr 500 505 510 515 ttg actttg gag aag ttc ctg gtc att gtc ttc ccc ttc agt aac att 1699 Leu Thr LeuGlu Lys Phe Leu Val Ile Val Phe Pro Phe Ser Asn Ile 520 525 530 cga cctgga aaa cgg cag acc tca gtc atc ctc att tgc atc tgg atg 1747 Arg Pro GlyLys Arg Gln Thr Ser Val Ile Leu Ile Cys Ile Trp Met 535 540 545 gcg ggattt tta ata gct gta att cca ttt tgg aat aag gat tat ttt 1795 Ala Gly PheLeu Ile Ala Val Ile Pro Phe Trp Asn Lys Asp Tyr Phe 550 555 560 gga aacttt tat ggg aaa aat gga gta tgt ttc cca ctt tat tat gac 1843 Gly Asn PheTyr Gly Lys Asn Gly Val Cys Phe Pro Leu Tyr Tyr Asp 565 570 575 caa acagaa gat att gga agc aaa ggg tat tct ctt gga att ttc cta 1891 Gln Thr GluAsp Ile Gly Ser Lys Gly Tyr Ser Leu Gly Ile Phe Leu 580 585 590 595 ggtgtg aac ttg ctg gct ttt ctc atc att gtg ttt tcc tat att act 1939 Gly ValAsn Leu Leu Ala Phe Leu Ile Ile Val Phe Ser Tyr Ile Thr 600 605 610 atgttc tgt tcc att caa aaa acc gcc ttg cag acc aca gaa gta agg 1987 Met PheCys Ser Ile Gln Lys Thr Ala Leu Gln Thr Thr Glu Val Arg 615 620 625 aattgt ttt gga aga gag gtg gct gtt gca aat cgt ttc ttt ttt ata 2035 Asn CysPhe Gly Arg Glu Val Ala Val Ala Asn Arg Phe Phe Phe Ile 630 635 640 gtgttc tct gat gcc atc tgc tgg att cct gta ttt gta gtt aaa atc 2083 Val PheSer Asp Ala Ile Cys Trp Ile Pro Val Phe Val Val Lys Ile 645 650 655 ctttcc ctc ttc cgg gtg gaa ata cca gac aca atg act tcc tgg ata 2131 Leu SerLeu Phe Arg Val Glu Ile Pro Asp Thr Met Thr Ser Trp Ile 660 665 670 675gtg att ttt ttc ctt cca gtt aac agt gct ttg aat cca atc ctc tat 2179 ValIle Phe Phe Leu Pro Val Asn Ser Ala Leu Asn Pro Ile Leu Tyr 680 685 690act ctc aca acc aac ttt ttt aag gac aag ttg aaa cag ctg ctg cac 2227 ThrLeu Thr Thr Asn Phe Phe Lys Asp Lys Leu Lys Gln Leu Leu His 695 700 705aaa cat cag agg aaa tca att ttc aaa att aaa aaa aaa agt tta tct 2275 LysHis Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys Lys Ser Leu Ser 710 715 720aca tcc att gtg tgg ata gag gac tcc tct tcc ctg aaa ctt ggg gtt 2323 ThrSer Ile Val Trp Ile Glu Asp Ser Ser Ser Leu Lys Leu Gly Val 725 730 735ttg aac aaa ata aca ctt gga gac agt ata atg aaa cca gtt tcc t 2369 LeuAsn Lys Ile Thr Leu Gly Asp Ser Ile Met Lys Pro Val Ser 740 745 750agcaatcatt ttggatcact ggactttcag tggactacct aaaacagggg acagcttttg 2429gaagatgaca tctgcaatgc ttttcatctt taccaacggc aagcctttct gcacagagag 2489cacagcagaa tggctcctgt cactgcattc caatggcagc tgtactatct accaaccatg 2549ctgaggacag caccaaaggt tcctctcctc accccacatg cctgaaaagc acatgtgaat 2609tcgtgtatag tgggctgagg tgcagctgat ctctagctaa tcaacacaac ccaccaacaa 2669atgaccacag gttggcactg tgtggtcttt cacatcgggt tgcactgtcc atgaaataga 2729aacactcaca acatctgatt ccagtgtggc cataataaca gaaatctaac aactctttcc 2789ttgccttttc aatatcaaat aaaaccatca gcatcctgct ggattgata 2838 2 754 PRT H.sapiens 2 Met Ile Val Phe Leu Val Phe Lys His Leu Phe Ser Leu Arg LeuIle 1 5 10 15 Thr Met Phe Phe Leu Leu His Phe Ile Val Leu Ile Asn ValLys Asp 20 25 30 Phe Ala Leu Thr Gln Gly Ser Met Ile Thr Pro Ser Cys GlnLys Gly 35 40 45 Tyr Phe Pro Cys Gly Asn Leu Thr Lys Cys Leu Pro Arg AlaPhe His 50 55 60 Cys Asp Gly Lys Asp Asp Cys Gly Asn Gly Ala Asp Glu GluAsn Cys 65 70 75 80 Gly Asp Thr Ser Gly Trp Ala Thr Ile Phe Gly Thr ValHis Gly Asn 85 90 95 Ala Asn Ser Val Ala Leu Thr Gln Glu Cys Phe Leu LysGln Tyr Pro 100 105 110 Gln Cys Cys Asp Cys Lys Glu Thr Glu Leu Glu CysVal Asn Gly Asp 115 120 125 Leu Lys Ser Val Pro Met Ile Ser Asn Asn ValThr Leu Leu Ser Leu 130 135 140 Lys Lys Asn Lys Ile His Ser Leu Pro AspLys Val Phe Ile Lys Tyr 145 150 155 160 Thr Lys Leu Lys Lys Ile Phe LeuGln His Asn Cys Ile Arg His Ile 165 170 175 Ser Arg Lys Ala Phe Phe GlyLeu Cys Asn Leu Gln Ile Leu Tyr Leu 180 185 190 Asn His Asn Cys Ile ThrThr Leu Arg Pro Gly Ile Phe Lys Asp Leu 195 200 205 His Gln Leu Thr TrpLeu Ile Leu Asp Asp Asn Pro Ile Thr Arg Ile 210 215 220 Ser Gln Arg LeuPhe Thr Gly Leu Asn Ser Leu Phe Phe Leu Ser Met 225 230 235 240 Val AsnAsn Tyr Leu Glu Ala Leu Pro Lys Gln Met Cys Ala Gln Met 245 250 255 ProGln Leu Asn Trp Val Asp Leu Glu Gly Asn Arg Ile Lys Tyr Leu 260 265 270Thr Asn Ser Thr Phe Leu Ser Cys Asp Ser Leu Thr Val Leu Phe Leu 275 280285 Pro Arg Asn Gln Ile Gly Phe Val Pro Glu Lys Thr Phe Ser Ser Leu 290295 300 Lys Asn Leu Gly Glu Leu Asp Leu Ser Ser Asn Thr Ile Thr Glu Leu305 310 315 320 Ser Pro His Leu Phe Lys Asp Leu Lys Leu Leu Gln Lys LeuAsn Leu 325 330 335 Ser Ser Asn Pro Leu Met Tyr Leu His Lys Asn Gln PheGlu Ser Leu 340 345 350 Lys Gln Leu Gln Ser Leu Asp Leu Glu Arg Ile GluIle Pro Asn Ile 355 360 365 Asn Thr Arg Met Phe Gln Pro Met Lys Asn LeuSer His Ile Tyr Phe 370 375 380 Lys Asn Phe Arg Tyr Cys Ser Tyr Ala ProHis Val Arg Ile Cys Met 385 390 395 400 Pro Leu Thr Asp Gly Ile Ser SerPhe Glu Asp Leu Leu Ala Asn Asn 405 410 415 Ile Leu Arg Ile Phe Val TrpVal Ile Ala Phe Ile Thr Cys Phe Gly 420 425 430 Asn Leu Phe Val Ile GlyMet Arg Ser Phe Ile Lys Ala Glu Asn Thr 435 440 445 Thr His Ala Met SerIle Lys Ile Leu Cys Cys Ala Asp Cys Leu Met 450 455 460 Gly Val Tyr LeuPhe Phe Val Gly Ile Phe Asp Ile Lys Tyr Arg Gly 465 470 475 480 Gln TyrGln Lys Tyr Ala Leu Leu Trp Met Glu Ser Val Gln Cys Arg 485 490 495 LeuMet Gly Phe Leu Ala Met Leu Ser Thr Glu Val Ser Val Leu Leu 500 505 510Leu Thr Tyr Leu Thr Leu Glu Lys Phe Leu Val Ile Val Phe Pro Phe 515 520525 Ser Asn Ile Arg Pro Gly Lys Arg Gln Thr Ser Val Ile Leu Ile Cys 530535 540 Ile Trp Met Ala Gly Phe Leu Ile Ala Val Ile Pro Phe Trp Asn Lys545 550 555 560 Asp Tyr Phe Gly Asn Phe Tyr Gly Lys Asn Gly Val Cys PhePro Leu 565 570 575 Tyr Tyr Asp Gln Thr Glu Asp Ile Gly Ser Lys Gly TyrSer Leu Gly 580 585 590 Ile Phe Leu Gly Val Asn Leu Leu Ala Phe Leu IleIle Val Phe Ser 595 600 605 Tyr Ile Thr Met Phe Cys Ser Ile Gln Lys ThrAla Leu Gln Thr Thr 610 615 620 Glu Val Arg Asn Cys Phe Gly Arg Glu ValAla Val Ala Asn Arg Phe 625 630 635 640 Phe Phe Ile Val Phe Ser Asp AlaIle Cys Trp Ile Pro Val Phe Val 645 650 655 Val Lys Ile Leu Ser Leu PheArg Val Glu Ile Pro Asp Thr Met Thr 660 665 670 Ser Trp Ile Val Ile PhePhe Leu Pro Val Asn Ser Ala Leu Asn Pro 675 680 685 Ile Leu Tyr Thr LeuThr Thr Asn Phe Phe Lys Asp Lys Leu Lys Gln 690 695 700 Leu Leu His LysHis Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys Lys 705 710 715 720 Ser LeuSer Thr Ser Ile Val Trp Ile Glu Asp Ser Ser Ser Leu Lys 725 730 735 LeuGly Val Leu Asn Lys Ile Thr Leu Gly Asp Ser Ile Met Lys Pro 740 745 750Val Ser 3 1115 PRT H. sapiens 3 Met Ala Thr Met Ser Gly Thr Thr Ile ValCys Leu Ile Tyr Leu Thr 1 5 10 15 Thr Met Leu Gly Asn Ser Gln Gly ValAsn Leu Lys Ile Glu Ser Pro 20 25 30 Ser Pro Pro Thr Leu Cys Ser Val GluGly Thr Phe His Cys Asp Asp 35 40 45 Gly Met Leu Gln Cys Val Leu Met GlySer Lys Cys Asp Gly Val Ser 50 55 60 Asp Cys Glu Asn Gly Met Asp Glu SerVal Glu Thr Cys Gly Cys Leu 65 70 75 80 Gln Ser Glu Phe Gln Cys Asn HisThr Thr Cys Ile Asp Lys Ile Leu 85 90 95 Arg Cys Asp Arg Asn Asp Asp CysSer Asn Gly Leu Asp Glu Arg Glu 100 105 110 Cys Asp Ile Tyr Ile Cys ProLeu Gly Thr His Val Lys Trp His Asn 115 120 125 His Phe Cys Val Pro ArgAsp Lys Gln Cys Asp Phe Leu Asp Asp Cys 130 135 140 Gly Asp Asn Ser AspGlu Lys Ile Cys Glu Arg Arg Glu Cys Val Ala 145 150 155 160 Thr Glu PheLys Cys Asn Asn Ser Gln Cys Val Ala Phe Gly Asn Leu 165 170 175 Cys AspGly Leu Val Asp Cys Val Asp Gly Ser Asp Glu Asp Gln Val 180 185 190 AlaCys Asp Ser Asp Lys Tyr Phe Gln Cys Ala Glu Gly Ser Leu Ile 195 200 205Lys Lys Glu Phe Val Cys Asp Gly Trp Val Asp Cys Lys Leu Thr Phe 210 215220 Ala Asp Glu Leu Asn Cys Lys Leu Cys Asp Glu Asp Asp Phe Arg Cys 225230 235 240 Ser Asp Thr Arg Cys Ile Gln Lys Ser Asn Val Cys Asp Gly TyrCys 245 250 255 Asp Cys Lys Thr Cys Asp Asp Glu Glu Val Cys Ala Asn AsnThr Tyr 260 265 270 Gly Cys Pro Met Asp Thr Lys Tyr Met Cys Arg Ser IleTyr Gly Glu 275 280 285 Pro Arg Cys Ile Asp Lys Asp Asn Val Cys Asn MetIle Asn Asp Cys 290 295 300 Arg Asp Gly Asn Val Gly Thr Asp Glu Tyr TyrCys Ser Asn Asp Ser 305 310 315 320 Glu Cys Lys Asn Phe Gln Ala Ala MetGly Phe Phe Tyr Cys Pro Glu 325 330 335 Glu Arg Cys Leu Ala Lys His LeuTyr Cys Asp Leu His Pro Asp Cys 340 345 350 Ile Asn Gly Glu Asp Glu GlnSer Cys Leu Ala Pro Pro Lys Cys Ser 355 360 365 Gln Asp Glu Phe Gln CysHis His Gly Lys Cys Ile Pro Ile Ser Lys 370 375 380 Arg Cys Asp Ser ValHis Asp Cys Val Asp Trp Ser Asp Glu Met Asn 385 390 395 400 Cys Glu AsnHis Gln Cys Ala Ala Asn Met Lys Ser Cys Leu Ser Gly 405 410 415 His CysIle Glu Glu His Lys Trp Cys Asn Phe His Arg Glu Cys Pro 420 425 430 AspGly Ser Asp Glu Lys Asp Cys Asp Pro Arg Pro Val Cys Glu Ala 435 440 445Asn Gln Phe Arg Cys Lys Asn Gly Gln Cys Ile Asp Pro Leu Gln Val 450 455460 Cys Val Lys Gly Asp Lys Tyr Asp Gly Cys Ala Asp Gln Ser His Leu 465470 475 480 Ile Asn Cys Ser Gln His Ile Cys Leu Glu Gly Gln Phe Arg CysArg 485 490 495 Lys Ser Phe Cys Ile Asn Gln Thr Lys Val Cys Asp Gly ThrVal Asp 500 505 510 Cys Leu Gln Gly Met Trp Asp Glu Asn Asn Cys Arg TyrTrp Cys Pro 515 520 525 His Gly Gln Ala Ile Cys Gln Cys Glu Gly Val ThrMet Asp Cys Thr 530 535 540 Gly Gln Lys Leu Lys Glu Met Pro Val Gln GlnMet Glu Glu Asp Leu 545 550 555 560 Ser Lys Leu Met Ile Gly Asp Asn LeuLeu Asn Leu Thr Ser Thr Thr 565 570 575 Phe Ser Ala Thr Tyr Tyr Asp LysVal Thr Tyr Leu Asp Leu Ser Arg 580 585 590 Asn His Leu Thr Glu Ile ProIle Tyr Ser Phe Gln Asn Met Trp Lys 595 600 605 Leu Thr His Leu Asn LeuAla Asp Asn Asn Ile Thr Ser Leu Lys Asn 610 615 620 Gly Ser Leu Leu GlyLeu Ser Asn Leu Lys Gln Leu His Ile Asn Gly 625 630 635 640 Asn Lys IleGlu Thr Ile Glu Glu Asp Thr Phe Ser Ser Met Ile His 645 650 655 Leu ThrVal Leu Asp Leu Ser Asn Gln Arg Leu Thr His Val Tyr Lys 660 665 670 AsnMet Phe Lys Gly Leu Lys Gln Ile Thr Val Leu Asn Ile Ser Arg 675 680 685Asn Gln Ile Asn Ser Ile Asp Asn Gly Ala Phe Asn Asn Leu Ala Asn 690 695700 Val Arg Leu Ile Asp Leu Ser Gly Asn Val Ile Lys Asp Ile Gly Gln 705710 715 720 Lys Val Phe Met Gly Leu Pro Arg Leu Val Glu Leu Lys Thr AspSer 725 730 735 Tyr Arg Phe Cys Cys Leu Ala Pro Glu Gly Val Lys Cys SerPro Lys 740 745 750 Gln Asp Glu Phe Ser Ser Cys Glu Asp Leu Met Ser AsnHis Val Leu 755 760 765 Arg Val Ser Ile Trp Val Leu Gly Val Ile Ala LeuVal Gly Asn Phe 770 775 780 Val Val Ile Phe Trp Arg Val Arg Asp Phe ArgGly Gly Lys Val His 785 790 795 800 Ser Phe Leu Ile Thr Asn Leu Ala IleGly Asp Phe Leu Met Gly Val 805 810 815 Tyr Leu Leu Ile Ile Ala Thr AlaAsp Thr Tyr Tyr Arg Gly Val Tyr 820 825 830 Ile Ser His Asp Glu Asn TrpLys Gln Ser Gly Leu Cys Gln Phe Ala 835 840 845 Gly Phe Val Ser Thr PheSer Ser Glu Leu Ser Val Leu Thr Leu Ser 850 855 860 Thr Ile Thr Leu AspArg Leu Ile Cys Ile Leu Phe Pro Leu Arg Arg 865 870 875 880 Thr Arg LeuGly Leu Arg Gln Ala Ile Ile Val Met Ser Cys Ile Trp 885 890 895 Val LeuVal Phe Leu Leu Ala Val Leu Pro Leu Leu Gly Phe Ser Tyr 900 905 910 PheGlu Asn Phe Tyr Gly Arg Ser Gly Val Cys Leu Ala Leu His Val 915 920 925Thr Pro Asp Arg Arg Pro Gly Trp Glu Tyr Ser Val Gly Val Phe Ile 930 935940 Leu Leu Asn Leu Leu Ser Phe Val Leu Ile Ala Ser Ser Tyr Leu Trp 945950 955 960 Met Phe Ser Val Ala Lys Lys Thr Arg Ser Ala Val Arg Thr AlaGlu 965 970 975 Ser Lys Asn Asp Asn Ala Met Ala Arg Arg Met Thr Leu IleVal Met 980 985 990 Thr Asp Phe Cys Cys Trp Val Pro Ile Ile Val Leu GlyPhe Val Ser 995 1000 1005 Leu Ala Gly Ala Arg Ala Asp Asp Gln Val TyrAla Trp Ile Ala Val 1010 1015 1020 Phe Val Leu Pro Leu Asn Ser Ala ThrAsn Pro Val Ile Tyr Thr Leu 1025 1030 1035 1040 Ser Thr Ala Pro Phe LeuGly Asn Val Arg Lys Arg Ala Asn Arg Phe 1045 1050 1055 Arg Lys Ser PheIle His Ser Phe Thr Gly Asp Thr Lys His Ser Tyr 1060 1065 1070 Val AspAsp Gly Thr Thr His Ser Tyr Cys Glu Lys Lys Ser Pro Tyr 1075 1080 1085Arg Gln Leu Glu Leu Lys Arg Leu Arg Ser Leu Asn Ser Ser Pro Pro 10901095 1100 Met Tyr Tyr Asn Thr Glu Leu His Ser Asp Ser 1105 1110 1115 4722 PRT H. sapiens 4 Lys Cys Pro Gly Gly Tyr Phe His Cys Asn Thr Thr AlaGln Cys Val 1 5 10 15 Pro Gln Arg Ala Asn Cys Asp Gly Ser Val Asp CysAsp Asp Ala Ser 20 25 30 Asp Glu Val Asn Cys Val Asn Glu Val Asp Ala LysTyr Trp Asp His 35 40 45 Leu Tyr Arg Lys Gln Pro Phe Gly Arg His Asp AsnLeu Arg Ile Gly 50 55 60 Glu Cys Leu Trp Pro Asn Glu Asn Phe Ser Cys ProCys Arg Gly Asp 65 70 75 80 Glu Ile Leu Cys Arg Phe Gln Gln Leu Thr AspIle Pro Glu Arg Leu 85 90 95 Pro Gln His Asp Leu Ala Thr Leu Asp Leu ThrGly Asn Asn Phe Glu 100 105 110 Thr Ile His Glu Thr Phe Phe Ser Glu LeuPro Asp Val Asp Ser Leu 115 120 125 Val Leu Lys Phe Cys Ser Ile Arg GluIle Ala Ser His Ala Phe Asp 130 135 140 Arg Leu Ala Asp Asn Pro Leu ArgThr Leu Tyr Met Asp Asp Asn Lys 145 150 155 160 Leu Pro His Leu Pro GluHis Phe Phe Pro Glu Gly Asn Gln Leu Ser 165 170 175 Ile Leu Ile Leu AlaArg Asn His Leu His His Leu Lys Arg Ser Asp 180 185 190 Phe Leu Asn LeuGln Lys Leu Gln Glu Leu Asp Leu Arg Gly Asn Arg 195 200 205 Ile Gly AsnPhe Glu Ala Glu Val Phe Ala Arg Leu Pro Asn Leu Glu 210 215 220 Val LeuTyr Leu Asn Glu Asn His Leu Lys Arg Leu Asp Pro Asp Arg 225 230 235 240Phe Pro Arg Thr Leu Leu Asn Leu His Thr Leu Ser Leu Ala Tyr Asn 245 250255 Gln Ile Glu Asp Ile Ala Ala Asn Thr Phe Pro Phe Pro Arg Leu Arg 260265 270 Tyr Leu Phe Leu Ala Gly Asn Arg Leu Ser His Ile Arg Asp Glu Thr275 280 285 Phe Cys Asn Leu Ser Asn Leu Gln Gly Leu His Leu Asn Glu AsnArg 290 295 300 Ile Glu Gly Phe Asp Leu Glu Ala Phe Ala Cys Leu Lys AsnLeu Thr 305 310 315 320 Ser Leu Leu Leu Thr Gly Asn Arg Phe Gln Thr LeuAsp Ser Arg Val 325 330 335 Leu Lys Asn Leu Ser Ser Leu Asp Tyr Ile TyrPhe Ser Trp Phe His 340 345 350 Leu Cys Ser Ala Ala Met Asn Val Arg ValCys Asp Pro His Gly Asp 355 360 365 Gly Ile Ser Ser Lys Leu His Leu LeuAsp Asn Gln Ile Leu Arg Gly 370 375 380 Ser Val Trp Val Met Ala Ser IleAla Val Val Gly Asn Leu Leu Val 385 390 395 400 Leu Leu Gly Arg Tyr PheTyr Lys Ser Arg Ser Asn Val Glu His Ser 405 410 415 Leu Tyr Leu Arg HisLeu Ala Ala Ser Asp Phe Leu Met Gly Ile Tyr 420 425 430 Leu Thr Leu IleAla Cys Ala Asp Ile Ser Phe Arg Gly Glu Tyr Ile 435 440 445 Lys Tyr GluGlu Thr Trp Arg His Ser Gly Val Cys Ala Phe Val Gly 450 455 460 Phe LeuSer Thr Phe Ser Cys Gln Ser Ser Thr Leu Leu Leu Thr Leu 465 470 475 480Val Thr Trp Asp Arg Leu Met Ser Val Thr Arg Pro Leu Lys Pro Arg 485 490495 Asp Thr Glu Lys Val Arg Ile Val Leu Arg Leu Leu Leu Leu Trp Gly 500505 510 Ile Ser Phe Gly Leu Ala Ala Ala Pro Leu Leu Pro Asn Pro Tyr Phe515 520 525 Gly Ser His Phe Tyr Gly Asn Asn Gly Val Cys Leu Ser Leu HisIle 530 535 540 His Asp Pro Tyr Ala Lys Gly Trp Glu Tyr Ser Ala Leu LeuPhe Ile 545 550 555 560 Leu Val Asn Thr Leu Ser Leu Ile Phe Ile Leu PheSer Tyr Ile Arg 565 570 575 Met Leu Gln Ala Ile Arg Asp Ser Gly Gly GlyMet Arg Ser Thr His 580 585 590 Ser Gly Arg Glu Asn Val Val Ala Thr ArgPhe Ala Ile Ile Val Thr 595 600 605 Thr Asp Cys Ala Cys Trp Leu Pro IleIle Val Val Lys Leu Ala Ala 610 615 620 Leu Ser Gly Cys Glu Ile Ser ProAsp Leu Tyr Ala Trp Leu Ala Val 625 630 635 640 Leu Val Leu Pro Val AsnSer Ala Leu Asn Pro Val Leu Tyr Thr Leu 645 650 655 Thr Thr Ala Ala PheLys Gln Gln Leu Arg Arg Tyr Cys His Thr Leu 660 665 670 Pro Ser Cys SerLeu Val Asn Asn Glu Thr Arg Ser Gln Thr Gln Thr 675 680 685 Ala Tyr GluSer Gly Leu Ser Val Ser Leu Ala His Leu Gly Gly Gly 690 695 700 Val GlyGly Gly Ser Gly Arg Lys Arg Met Ser His Arg Gln Met Ser 705 710 715 720Tyr Leu

What is claimed is:
 1. A composition comprising an LGR8 polypeptide,comprising at least 18 contiguous amino acids of the sequence set forthin SEQ ID NO:2.
 2. A composition according to claim 1, wherein said LGR8polypeptide is a soluble fragment of LGR8.
 3. A composition according toclaim 1, wherein said LGR8 polypeptide comprises a mutation that confersa gain of function.
 4. A composition comprising an LGR7 polypeptide,wherein said polypeptide is a soluble fragment of LGR7.
 5. Thecomposition according to claim 2, wherein said composition furthercomprises a pharmaceutically acceptable carrier.
 6. The compositionaccording to claim 4, wherein said composition further comprises apharmaceutically acceptable carrier.
 7. A method of inhibiting prematurelabor, the method comprising administering a patient suffering frompremature labor the composition according to claim
 2. 8. A method ofinhibiting premature labor, the method comprising administering apatient suffering from premature labor the composition according toclaim
 5. 9. An isolated nucleic acid molecule comprising a cDNA sequenceencoding a mammalian LGR8 polypeptide that will hybridize understringent conditions of 50° C. or higher in the presence of 0.1×SSC tothe sequence set forth in SEQ ID NO:1, or encodes the peptide of SEQ IDNO:2.
 10. An antibody that specifically recognizes a relaxin receptor.11. A non-human transgenic animal model for relaxin receptor genefunction wherein said transgenic animal comprises an introducedalteration in a LGR7 or LGR8 gene.
 12. A method of screening forbiologically active agents that modulate relaxin function, the methodcomprising: combining a candidate biologically active agent with any oneof: (a) an LGR7 or LGR8 polypeptide; (b) a cell comprising a nucleicacid encoding an LGR7 or LGR8 polypeptide; or (c) a non-human transgenicanimal model for relaxin receptor gene function comprising one of: (i) aknockout of an LGR7 or LGR8 gene; (ii) an exogenous and stablytransmitted LGR7 or LGR8 gene; and determining the effect of said agenton relaxin function.
 13. A method of screening for biologically activeagents that modulate InsL3 function, the method comprising: combining acandidate biologically active agent with any one of: (a) an LGR8polypeptide; (b) a cell comprising a nucleic acid encoding an LGR8polypeptide; or (c) a non-human transgenic animal model for relaxinreceptor gene function comprising one of: (i) a knockout of an LGR8gene; (ii) an exogenous and stably transmitted LGR8 gene; anddetermining the effect of said agent on LGR8 function.
 14. A method ofdesigning biologically active agents that modulate LGR7 or LGR8function, the method comprising: determining the binding sites betweenLGR8 or LGR7 and a cognate ligand; designing a pharmacomimetic moleculethat mimics the binding site of either said LGR8 or LGR7; or the bindingsite of said cognate ligand.
 15. The method according to claim 13,wherein said cognate ligand is relaxin or InsL3.
 16. A method for thetreatment of cryptorchidism, the method comprising: administering to anindividual suffering from said cryptorchidism an agonist of LGR8 in apharmaceutically effective dose.
 17. A method for the treatment ofscleroderma, the method comprising: administering to an individualsuffering from said scleroderma an agonist of LGR7 or LGR8 in apharmaceutically effective dose.
 18. A method for the induction oflabor, the method comprising: administering to an individual for whichinduction of labor is desired, an agonist of LGR7 or LGR8 in apharmaceutically effective dose.
 19. A method for the diagnosis of agenetic condition associated LGR8 or LGR7, the method comprising:analyzing a sample of a patient suspected of said genetic condition forexpression or sequence of LGR7 or LGR8, wherein an alteration inexpression or sequence as compared to a normal sample is indicative ofsaid genetic condition.
 20. The method according to claim 19, whereinsaid condition is cryptorchidism.